High affinity antibody antagonists of interleukin-13 receptor alpha 1

ABSTRACT

High affinity antibody antagonists of human interleukin-13 receptor alpha 1 are disclosed. The antibody molecules are effective in the inhibition of IL-13Rα1-mediated activities and, accordingly, present desirable antagonists for the use in the treatment of conditions associated with hIL-13Rα1 activity. The present invention also discloses nucleic acid encoding said antibody molecules, vectors, host cells, and compositions comprising the antibody molecules. Methods of using the antibody molecules for inhibiting or antagonizing IL-13Rα1-mediated activities are also disclosed.

This application is a continuation of U.S. Ser. No. 11/875,017 filedOct. 19, 2007, now U.S. Pat. No. 7,754,213 which claims benefit ofpriority to U.S. Provisional Patent Application Ser. No. 60/852,884,filed Oct. 19, 2006, each of which are herein incorporated by referencein their entireties.

BACKGROUND OF THE INVENTION

Data from human studies and experimental animal models stronglyimplicate Th2-derived cytokines as contributing to atopic asthma, withinterleukin-4 (IL-4) and interleukin-13 (IL-13; see, e.g., Minty et al.,1993 Nature 362:248-250; McKenzie et al., 1993 Proc. Natl. Acad. Sci.USA 90:3735-3739; Acc. Nos: U31120 and L13029 (human) andNM_(—)001032929 (Macaca mulatta)) playing the most central role. Thesetwo cytokines have significant structural similarities and share certainreceptor components. The receptor that IL-4 and IL-13 share is a dualIL-4R/IL-13 receptor (or type II receptor) which binds both IL-4 andIL-13. This receptor is composed of the IL-4Rα chain (see, e.g., Idzerdaet al., 1990 J. Exp. Med. 171:861-873) and the IL-13Rα1 chain (see,e.g., Hilton et al., 1996 Proc. Natl. Acad. Sci. USA 93:497-501; Aman etal., 1996 J. Biol. Chem. 271:29265-29270; Miloux et al., 1997 FEBS Lett.401:163-166; Acc. Nos: U62858 and CAA70508 (human) and AAP78901 (Macacafascicularis)). The dual IL-4R/IL-13R receptor is expressed onhematopoietic and non-hematopoietic cells, including lung epithelial andsmooth muscle cells. Both IL-4 and IL-13, additionally, each have onereceptor that recognizes them to the exclusion of the other. Forinstance, IL-4 receptor (IL-4R) type I, composed of the IL-4Rα chain andthe common gamma chain (γc), specifically binds IL-4. IL-4R type I isexpressed exclusively on cells of hematopoietic origin. The receptorspecific for IL-13, IL-13Rα2, binds IL-13 with high affinity, butapparently does not transduce signals. Rather, the receptor acts as adecoy to attenuate the response to IL-13.

IL-13 and IL-4 carry out a number of functions and both regulate anumber of functions related to the allergic phenotype, such as isotypeclass switching to IgE in B-cells, activation of mast cells andeosinophils, up-regulation of vascular cell adhesion molecule-1 (VCAM-1)on endothelial cells, and production of chemokines such as eotaxins,thymus and activation-regulated chemokine (TARC), and macrophage-derivedchemokine (MDC).

IL-4 and IL-13, though, have many distinct functions in vitro and invivo owing to differences in their receptor complexes. For instance,sequestration of IL-13, but not IL-4, has been shown to prevent airwayhyperreactivity and reduce mucous production in mouse asthma models.This correlation between IL-13 and the asthmatic response has beenfurther supported by other studies; see, e.g., Hershey et al., 2003 J.Allergy Clin. Immunol. 111(4):677-690; Grunig et al., 1998 Science282(5397):2261-2263; Mattes et al., 2001 J. Immunol. 167(3):1683-1692;and Fulkerson et al., 2006 Am. J. Respir. Cell. Mol. Biol. 35(3)337-346.Delivery of IL-13 to the lung, for example, has been found to besufficient to induce the entire asthma-like phenotype in mice. Treatedanimals develop airway hyperreactivity, eosinophil-rich cellinflammation, goblet cell hyperplasia with associated mucousoverproduction, and subepithelial fibrosis; see, e.g., Wills-Karp etal., 1998 Science 282(5397): 2258-2261; Reiman et al., 2006 Infect.Immun. 74(3): 1471-1479; and Kaviratne et al., 2004 J. Immunol.173(6):4020-4029. Expression of IL-13 has, furthermore, been reported tobe elevated in the lungs of human asthmatics. In addition, severalgroups have reported associations of polymorphisms in the IL-13 genewith an increased risk of allergic traits and asthma symptoms. Some ofthese polymorphisms have been shown to be correlated with increasedexpression of IL-13; see, e.g., Huang et al., 1995 J. Immunol.155(5)2688-2694; Naseer et al., 1997 Am. J. Respir. Crit. Care Med.155(3):845-851; Vladich et al., 2005 J. Clin. Invest. 115(3):747-754;Chen et al., 2004J. Allergy Clin. Immunol. 114(3):553-560; and Vercelliet al., 2002 Curr. Opin. Allergy Clin. Immunol. 2(5):389-393.

IL-13 has also been associated with various other conditions, includingbut not limited to various respiratory and allergy-mediated disorders,fibrosis, scleroderma, inflammatory bowel disease and certain cancers;see, e.g., Wynn, T. A., 2003 Annu. Rev. Immunol. 21:425-456; Terabe etal., 2000 Nat. Immunol. 1(6):515-520; Fuss et al., 2004 J. Clin. Invest.113(10):1490-1497; Simms et al., 2002 Curr. Opin. Rheumatol.14(6):717-722; and Hasegawa et al., 1997 J. Rheumatol. 24(2):328-332.

An antagonist of IL-13 would, therefore, be a highly attractive moleculefor use in the development of a treatment for IL-13-associateddisorders. An effective antibody antagonist would interfere with thebinding of IL-13 to IL-13R. An effective antibody antagonist to IL-13Rα1may also interfere with the binding of IL-13 and preventheterodimerization of IL-4Rα and IL-13Rα1. Such an antibody couldinhibit signaling of both IL-13 and IL-4 through the type II receptorwhile sparing IL-4 signaling through the type I receptor. Signalingthrough the type I receptor is essential in the induction phase of theimmune response during which Th2 cells differentiate. T cells do notexpress IL-13Rα1 so the type II receptor plays no role in Th2differentiation. Hence, an IL-13Rα1 antibody should not affect theoverall Th1/Th2 balance. Signaling through the type II IL-4/IL-13receptor is critical during the effector stage of the immune responseduring established allergic inflammation. Thus, blockade of the type IIreceptor should have a beneficial effect on many of the symptoms ofasthma and other IL-13R-mediated conditions and should, therefore, be aneffective disease modifying agent.

Antibodies against IL-13Rα1 (both monoclonal and polyclonal) have beendescribed in the art; see, e.g., WO 97/15663, WO 03/80675; WO 03/46009;WO 06/072564; Gauchat et al., 1998 Eur. J. Immunol. 28:4286-4298;Gauchat et al., 2000 Eur. J. Immunol. 30:3157-3164; Clement et al., 1997Cytokine 9(11):959 (Meeting Abstract); Ogata et al., 1998 J. Biol. Chem.273:9864-9871; Graber et al., 1998 Eur. J. Immunol. 28:4286-4298; C.Vermot-Desroches et al., 2000 Tissue Antigens 5(Supp. 1):52-53 (MeetingAbstract); Poudrier et al., 2000 Eur. J. Immunol. 30:3157-3164; Akaiwaet al., 2001 Cytokine 13:75-84; Cancino-Díaz at al., 2002 J. Invest.Dermatol. 119:1114-1120; and Krause et al., 2006 Mol. Immunol.43:1799-1807.

There is a need for an antibody with enhanced biological activity thatcould impact activities associated with the allergy and asthmaticresponse as well as other various conditions that have been attributedat least in part to an increased expression/functioning of IL-13Rα1.There is further a need for an antibody molecule with high affinity forIL-13Rα1 with low immunogenicity in humans. Accordingly, it would be ofgreat import to produce a therapeutic-based human antibody antagonist ofIL-13Rα1 that inhibits or antagonizes the activity of IL-13Rα1 and thecorresponding role IL-13Rα1 plays in various therapeutic conditions.

SUMMARY OF THE INVENTION

The present invention relates to high affinity antibody antagonists ofIL-13Rα1 and particularly human IL-13Rα1. Disclosed antibody moleculesselectively recognize IL-13Rα1, particularly human IL-13Rα1, exhibitingbinding to human IL-13Rα1 with a K_(D) of 5×10⁻⁹ or less, morepreferably 2×10⁻⁹ or less, and even more preferably, 1×10⁻⁹ or less.Specific antibody molecules in accordance herewith additionally, bindprimate IL-13Rα1 with high affinity, a desirable quality given theaccessibility of non-human primate models for predicting efficacy andsafety profiles in humans. Antibody molecules in accordance herewith areeffective in the inhibition of IL-13Rα1-mediated activities and, thus,are of import in the treatment of conditions associated therewith,including, but not limited to, asthma, allergy, allergic rhinitis,chronic sinusitis, hay fever, atopic dermatitis, chronic obstructivepulmonary disease (“COPD”), pulmonary fibrosis, esophageal eosinophilia,scleroderma, psoriasis, psoriatic arthritis, fibrosis, inflammatorybowel disease (particularly, ulcerative colitis), anaphylaxis, andcancer (particularly, Hodgkin's lymphoma, glioma, and renal carcinoma),and general Th2-mediated disorders/conditions. IL-13Rα1-specificantibodies also have utility for various diagnostic purposes in thedetection and quantification of IL-13Rα1.

The present invention provides, in one particular aspect, the isolatedantibody, 10G5, which very effectively antagonizes IL-13 functioningthrough IL-13Rα1. 10G5 exhibits inhibition of IL-13- and IL-4-inducedeotaxin release in NHDF cells, IL-13- and IL-4-induced STAT6phosphorylation in NHDF cells, and IL-13-stimulated release of TARC inblood or peripheral blood mononuclear cells (PBMCs). The presentinvention, thus, encompasses antibodies as produced by the hybridomacell line deposited as ATCC Deposit No. PTA-6933. The present inventionalso encompasses antibodies that compete for binding to hIL-13Rα1 withan antibody of PTA-6933. Particular embodiments of the present inventioninclude antibody molecules including heavy and/or light chain variableregion sequences of 10G5, as well as equivalents (characterized ashaving one or more conservative amino acid substitutions) or homologsthereof. Particular embodiments embrace isolated antibody molecules thathave the CDR domains disclosed herein or sets of heavy and/or lightchain CDR domains disclosed herein, or equivalents thereof,characterized as having one or more conservative amino acidsubstitutions. Specifically, antibody molecules of the invention have aheavy chain variable region with CDR1, CDR2, and CDR3 amino acidsequences as set forth in SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:121,respectively; and a light chain variable region with CDR1, CDR2, andCDR3 amino acid sequences as set forth in SEQ ID NO:84, SEQ ID NO:85,and SEQ ID NO:122. More particularly, antibody molecules of theinvention have a heavy chain variable region with an amino acid sequenceas set forth in SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO: 55, SEQ ID NO:59,SEQ ID NO:63 or SEQ ID NO:67; a light chain variable region with anamino acids sequence as set forth in SEQ ID NO:49, SEQ ID NO:71, SEQ IDNO:75 or SEQ ID NO:79; or a combination of the above-referenced heavychain and light chain variable regions.

As will be appreciated by those skilled in the art, fragments of anantibody that retain the ability to bind to hIL-13Rα1 may be insertedinto various frameworks, see, e.g., U.S. Pat. No. 6,818,418, andreferences contained therein, which discuss various scaffolds which maybe used to display antibody loops previously selected on the basis ofantigen binding. In addition, genes encoding for V_(L) and V_(H) can bejoined, using recombinant methods, for example using a synthetic linkerthat enables them to be made as a single protein chain in which theV_(L) and V_(H) regions pair to form monovalent molecules, otherwiseknown as single chain Fvs (ScFVs); see, e.g., Bird et al., 1988 Science242: 423-426, and Huston et al., 1988 Proc. Natl. Acad. Sci. USA85:5879-5883.

In another aspect, the present invention provides nucleic acid encodingthe disclosed antibody molecules. The present invention also providesnucleic acids encoding the variable heavy and light chains and selectcomponents thereof, particularly the disclosed respective CDR3 regions.In another aspect, the present invention provides vectors including saidnucleic acid. In another aspect, the present invention provides isolatedcell(s) harboring nucleic acid encoding the disclosed antibody moleculesand components thereof as described. In another aspect, the presentinvention provides isolated cell(s) comprising a polypeptide or vectorof the present invention.

In another aspect, the present invention provides a method of making anantibody molecule which selectively binds IL-13Rα1 (inclusive ofantibodies, antigen binding fragments, derivatives, chimeric molecules,fusions of any of the foregoing with another polypeptide, or alternativestructures/compositions incorporating any of the foregoing) of thepresent invention, which includes incubating a cell harboring nucleicacid encoding a heavy and/or a light chain (depending on the antibodymolecule being produced) under conditions that allow for the expressionand/or assembly of said heavy and/or light chains into the antibodymolecule, and isolating said antibody molecule from the cell. One ofskill in the art can obtain the antibody molecules disclosed hereinusing standard recombinant DNA techniques.

In another aspect, the present invention provides a method forantagonizing the activity or function of IL-13Rα1, be it signaling orother, which includes contacting a cell expressing IL-13Rα1 with anantibody molecule disclosed herein under conditions that allow saidantibody molecule to bind to IL-13Rα1. Specific embodiments of thepresent invention include such methods wherein the cell is a human cell.Antibody molecules in accordance herewith are effective in theinhibition of IL-13Rα1-mediated activities. Antibody molecules inaccordance with the present invention were found to effectively inhibiteotaxin release from normal human dermal fibroblast cells (hereinafter“NHDF” cells). Antibody molecules in accordance with the presentinvention were found to effectively inhibit IL-13- and IL-4-stimulatedSTAT6 phosphorylation in NHDF cells and found to effectively inhibit theIL-13-stimulated release of TARC (CCL17) in whole blood (human/rhesus).

In another aspect, the present invention provides a method ofantagonizing the activity of IL-13Rα1 in a subject exhibiting acondition associated with IL-13Rα1 activity (or a condition where thefunctioning of IL-13Rα1 is deemed not beneficial to the particularsubject), which involves administering to the subject a therapeuticallyeffective amount of an antibody molecule of the present invention. Inselect embodiments, the condition may be asthma, allergy, allergicrhinitis, chronic sinusitis, hay fever, atopic dermatitis, chronicobstructive pulmonary disease (“COPD”), pulmonary fibrosis, esophagealeosinophilia, psoriasis, psoriatic arthritis, fibrosis, scleroderma,inflammatory bowel disease (particularly, ulcerative colitis),anaphylaxis, and cancer (particularly, Hodgkin's lymphoma, glioma, andrenal carcinoma), and general Th2-mediated disorders/conditions. Inanother aspect, the present invention provides a pharmaceuticalcomposition or other composition including an antibody molecule of theinvention (or alternative antigen-binding structure or protein thatcomprises an IL-13Rα1-specific antigen binding portion disclosed herein)and a pharmaceutically acceptable carrier, excipient, diluent,stabilizer, buffer, or alternative designed to facilitate administrationof the antibody molecule in the desired amount to the treatedindividual.

Another aspect of the present invention concerns the identification of acritical contact point between the antibodies disclosed herein andhIL-13Rα1. This critical contact point was identified by the generationof a specific mutation that impacted the binding of the receptor by theantibody. More specifically, it was found that substitution of thephenylalanine residue at position 233 of SEQ ID NO:101 with an alanineresidue results in a loss of binding between the antibody and thereceptor. This is a very useful finding in characterizing 10G5 and itsderivatives. Peptides exploiting the narrow region surrounding aminoacid 233 will be useful in the generation of additional monoclonalantibodies with similar specificity. Accordingly, the present inventionencompasses isolated peptides including hIL-13R sequence with aPhe233Ala mutation, whether they be full-length or fragments thereofincluding the mutation. Additionally, the present invention alsoencompasses the use of said peptides in an assay to evaluate 10G5 or10G5 derivatives, identify antibodies with specificity for a similarepitope or, alternatively, produce antibodies with similar specificity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide and derived amino acid sequence of theheavy chain variable region of antibody 10G5. CDRs and nucleic acidencoding CDRs are underlined.

FIG. 2 illustrates the nucleotide and derived amino acid sequence of thelight chain variable region of antibody 10G5. CDRs and nucleic acidencoding CDRs are underlined.

FIG. 3 illustrates a genetic map of pFab3d.

FIGS. 4A and 4B illustrate heavy and light chain constructs, SEQ IDNOs:86 AND 87, respectively, of 10G5 Fab in pFab3d.

FIG. 5 illustrates binding of mutated Fabs from periplasmic preparationsto rhesus IL-13Rα1.

FIG. 6 illustrates ELISA analysis of binding of periplasmic preparationsof 10G5 mutants to human IL-13Rα1.

FIG. 7 illustrates ELISA analysis of binding of periplasmic preparationsof 10G5 mutants to human IL-13Rα1.

FIG. 8 illustrates Myc-capture ELISA data for light chain variantclones.

FIG. 9 illustrates the functioning of 10G5, 10G5-6, and 10G5-6 in a NHDFeotaxin release assay.

FIG. 10 illustrates the functioning of 10G5-6 in blocking IL-13- andIL-4-stimulated eotaxin release in NHDF cells.

FIG. 11 illustrates the functioning of 10G5 and 10G5H6 in anIL-13-induced STAT6 assay.

FIG. 12 illustrates the functioning of 10G5 and 10G5H6 in anIL-4-induced STAT6 assay.

FIG. 13 illustrates the functioning of 10G5-6 in blockingIL-13-stimulated release of TARC (CCL17) in whole human blood.

FIG. 14 illustrates the functioning of 10G5 and 10G5H6 in anIL-13-stimulated TARC release assay in whole human blood.

FIG. 15 illustrates the functioning of 10G5H6 and 10G5-6 in blockingrhesus IL-13-stimulated release of TARC in whole rhesus blood.

FIG. 16 illustrates a sequence comparison of the Fc domains of IgG1 (SEQID NO:97), IgG2 (SEQ ID NO:98), IgG4 (SEQ ID NO:99) and the IgG2m4 (SEQID NO:100) isotypes.

FIG. 17 illustrates inhibition of cell proliferation of Hodgkin'sDisease cell line L1236 by 10G5, 10G5H6, and 10G5-6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to high affinity antibody antagonists ofIL-13Rα1 and particularly human IL-13Rα1. Disclosed antibody moleculesselectively recognize and specifically bind to IL-13Rα1. In particularembodiments, the antibodies bind primate IL-13Rα1 (e.g., cynomolgusIL-13Rα1) with high affinity, a desirable quality given theaccessibility of non-human primate models for predicting efficacy andsafety profiles in humans. Use of the terms “selective” or “specific”herein refers to the fact that the disclosed antibody molecules do notshow significant binding to other than IL-13Rα1. The disclosed antibodymolecules bind to human IL-13Rα1 with a K_(D) of 5×10⁻⁹ or less, morepreferably 2×10⁻⁹ or less, and even more preferably, 1×10⁻⁹ or less.K_(D) refers to the dissociation constant obtained from the ratio ofK_(d) (the dissociation rate of a particular antibody-antigeninteraction) to K_(a) (the association rate of the particularantibody-antigen interaction), or K_(d)/K_(a) which is expressed as amolar concentration (M). K_(D) values can be determined using methodswell established in the art. A preferred method for determining theK_(D) of an antibody is by using surface plasmon resonance, for examplea biosensor system such as a BIACORE™ (Pharmacia AB Corporation) system.

Antibodies, as described herein, are particularly effective inantagonizing IL-13Rα1 function, or IL-13Rα1-mediated activity asreferred to herein. The language “IL-13Rα1-mediated” activity/functionis used herein to refer to any activity/function that requires, or isexacerbated or enhanced by, the function of IL-13Rα1. The disclosedantibody molecules have been shown to exhibit at least one of thefollowing functional properties: (i) inhibition of IL-13-induced eotaxinrelease in NHDF cells; (ii) inhibition of IL-4-induced eotaxin releasein NHDF cells; (iii) inhibition of IL-13-induced STAT6 phosphorylationin NHDF cells; (iv) inhibition of IL-4-induced STAT6 phosphorylation inNHDF cells; or (v) inhibition of IL-13-stimulated release of TARC inblood or PBMCs. Specific embodiments of the present invention provideantibody molecules that antagonize IL-13Rα1-mediated eotaxin releasefrom NHDF cells with an IC₅₀ of 1.0 μg/ml or less, more preferably, 0.5μg/ml or less, and more preferably yet, 0.1 μg/ml or less. Specificembodiments of the present invention provide antibody molecules thatantagonize IL-13Rα1-mediated STAT6 phosphorylation in NHDF cells.Specific embodiments of the present invention provide antibody moleculesthat antagonize IL-13Rα1-mediated TARC (CCL17) release in whole blood orPBMCs with an IC₅₀ of 1000 ng/ml or less, more preferably, 500 ng/ml orless, and more preferably yet, 250 ng/ml or less. The extent ofantagonism by any particular antibody can be measured quantitatively asthe IC₅₀ value in statistical comparison to a control, or via anyalternative method available in the art for assessing a negative effecton, or inhibition of, IL-13Rα1 function (i.e., any method capable ofassessing antagonism of IL-13Rα1 function).

“Antibody molecule” or “antibody”, as described herein, refers to animmunoglobulin-derived structure with selective binding to hIL-13Rα1including, but not limited to, a full length or whole antibody, anantigen binding fragment (a fragment derived, physically orconceptually, from an antibody structure), a derivative of any of theforegoing, a chimeric molecule, a fusion of any of the foregoing withanother polypeptide, or any alternative structure/composition whichincorporates any of the foregoing for purposes of selectivelybinding/inhibiting the function of IL-13Rα1. “Whole” antibodies or“full-length” antibodies refer to proteins that have two heavy (H) andtwo light (L) chains inter-connected by disulfide bonds which include:(1) in terms of the heavy chains, a variable region (abbreviated hereinas “V_(H)”) and a heavy chain constant region which has three domains,C_(H1), C_(H2), and C_(H3); and (2) in terms of the light chains, alight chain variable region (abbreviated herein as “V_(L)”) and a lightchain constant region which includes one domain, C_(L). “Isolated”, asused herein, describes a property as it pertains to the disclosedantibody molecules, nucleic acid or other that makes them different fromthat found in nature. The difference can be, for example, that they areof a different purity than that found in nature, or that they are of adifferent structure or form than that found in nature. A structure notfound in nature, for example, includes recombinant human immunoglobulinstructures including, but not limited to, recombinant humanimmunoglobulin structures with optimized complementarity determiningregions (CDRs). Other examples of structures not found in nature areantibody molecules or nucleic acid substantially free of other cellularmaterial. Isolated antibodies are generally free of other antibodieshaving different antigenic specificities (other than IL-13Rα1).

Antibody fragments and, more specifically, antigen binding fragments aremolecules possessing an antibody variable region or segment thereof(which includes one or more of the disclosed CDR 3 domains, heavy and/orlight), which confers selective binding to IL-13Rα1, and particularlyhuman IL-13Rα1 (hIL-13Rα1). Antibody fragments containing such anantibody variable region include, but are not limited to the followingantibody molecules: a Fab, a F(ab′)₂, a Fd, a Fv, a scFv, bispecificantibody molecules (i.e., antibody molecules including anIL-13Rα1-specific antibody or antigen binding fragment as disclosedherein linked to a second functional moiety having a different bindingspecificity than the antibody, including, without limitation, anotherpeptide or protein such as an antibody, or receptor ligand), abispecific single chain Fv dimer, an isolated CDR3, a minibody, a‘scAb’, a dAb fragment, a diabody, a triabody, a tetrabody, andartificial antibodies based upon protein scaffolds, including but notlimited to fibronectin type III polypeptide antibodies (see, e.g., U.S.Pat. No. 6,703,199, WO 02/32925 and WO 00/34784) or cytochrome B (see,e.g., Nygren et al., 1997 Curr. Opinion Struct. Biol. 7:463-469). Theantibody portions or binding fragments may be natural, or partly orwholly synthetically produced. Such antibody portions can be prepared byvarious means known by one of skill in the art, including, but notlimited to, conventional techniques, such as papain or pepsin digestion.

The present invention provides, in one particular aspect, isolatedantibody 10G5 which very effectively antagonizes IL-13 functioningthrough IL-13Rα1. 10G5 has exhibited inhibition of IL-13- andIL-4-induced eotaxin release in NHDF cells, IL-13- and IL-4-inducedSTAT6 phosphorylation in NHDF cells, and IL-13-stimulated release ofTARC in blood or peripheral blood mononuclear cells (PBMCs). The presentinvention, thus, encompasses antibodies as produced by the hybridomacell line deposited as ATCC Deposit No. PTA-6933. The present inventionalso encompasses antibody molecules that compete for binding tohIL-13Rα1 with an antibody of PTA-6933. Additional embodiments of thepresent invention are antibody molecules that compete for binding tohIL-13Rα1 with antibodies disclosed herein. Specific embodiments of thepresent invention provide isolated antibody molecules which inhibit thebinding of IL-13 to hIL-13Rα1.

Particular embodiments of the present invention include antibodymolecules having heavy and/or light chain variable region sequences of10G5, as well as equivalents (characterized as having one or moreconservative amino acid substitutions) or homologs thereof. Particularembodiments are isolated antibody molecules that include the CDR domainsdisclosed herein or sets of heavy and/or light chain CDR domainsdisclosed herein, or equivalents thereof, characterized as having one ormore conservative amino acid substitutions. Use of the terms “domain” or“region” herein simply refers to the respective portion of the antibodymolecule wherein the sequence or segment at issue will reside or, in thealternative, currently resides.

Table 1 provides a generalized outline of sequences embraced by thepresent invention.

TABLE 1 SEQ ID NO: DESCRIPTION SEQ ID NO: 1 HEAVY CHAIN CDR3 OPTIMIZEDVARIANT SEQ ID NO: 2 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 3HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 4 HEAVY CHAIN CDR3OPTIMIZED VARIANT SEQ ID NO: 5 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ IDNO: 6 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 7 HEAVY CHAIN CDR3OPTIMIZED VARIANT SEQ ID NO: 8 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ IDNO: 9 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 10 HEAVY CHAIN CDR3OPTIMIZED VARIANT SEQ ID NO: 11 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQID NO: 12 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 13 HEAVY CHAINCDR3 OPTIMIZED VARIANT SEQ ID NO: 14 HEAVY CHAIN CDR3 OPTIMIZED VARIANTSEQ ID NO: 15 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 16 HEAVYCHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 17 HEAVY CHAIN CDR3 OPTIMIZEDVARIANT SEQ ID NO: 18 HEAVY CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 19VARIANT SEQUENCE W/ OPTIMIZED CDR3 SEQ ID NO: 20 VARIANT SEQUENCE W/OPTIMIZED CDR3 SEQ ID NO: 21 VARIANT SEQUENCE W/ OPTIMIZED CDR3 SEQ IDNO: 22 VARIANT SEQUENCE W/ OPTIMIZED CDR3 SEQ ID NO: 23 VARIANT SEQUENCEW/ OPTIMIZED CDR3 SEQ ID NO: 24 VARIANT SEQUENCE W/ OPTIMIZED CDR3 SEQID NO: 25 VARIANT SEQUENCE W/ OPTIMIZED CDR3 SEQ ID NO: 26 HEAVY CHAINCDR3 OPTIMIZED VARIANT SEQ ID NO: 27 LIGHT CHAIN CDR3 OPTIMIZED VARIANTSEQ ID NO: 28 LIGHT CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 29 LIGHTCHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 30 LIGHT CHAIN CDR3 OPTIMIZEDVARIANT SEQ ID NO: 31 LIGHT CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 32LIGHT CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 33 LIGHT CHAIN CDR3OPTIMIZED VARIANT SEQ ID NO: 34 LIGHT CHAIN CDR3 OPTIMIZED VARIANT SEQID NO: 35 LIGHT CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 36 LIGHT CHAINCDR3 OPTIMIZED VARIANT SEQ ID NO: 37 LIGHT CHAIN CDR3 OPTIMIZED VARIANTSEQ ID NO: 38 LIGHT CHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 39 LIGHTCHAIN CDR3 OPTIMIZED VARIANT SEQ ID NO: 40 10G5 HEAVY CHAIN CDR3 SEQ IDNO: 41 10G5 LIGHT CHAIN CDR3 SEQ ID NO: 42 10G5 VH SEQ W/ LEADER, &ADDITIONAL CONSTANT REGION - NUCLEIC ACID SEQ ID NO: 43 10G5 VH SEQUENCEONLY - NUCLEIC ACID SEQ ID NO: 44 10G5 VH SEQ W/ LEADER, & ADDITIONALCONSTANT REGION - PROTEIN SEQ ID NO: 45 10G5 VH SEQUENCE ONLY - PROTEINSEQ ID NO: 46 10G5 VL SEQ W/ LEADER & ADDITIONAL CONSTANT REGION -NUCLEIC ACID SEQ ID NO: 47 10G5 VL SEQUENCE ONLY - NUCLEIC ACID SEQ IDNO: 48 10G5 VL SEQ W/ LEADER & ADDITIONAL CONSTANT REGION - PROTEIN SEQID NO: 49 10G5 VL SEQUENCE ONLY - PROTEIN SEQ ID NO: 50 10G5-1,3 HEAVYCHAIN* SEQ ID NO: 51 10G5-1,3 VH* SEQ ID NO: 52 10G5-1,3 HEAVY CHAINNUCLEIC ACID* SEQ ID NO: 53 10G5-1,3 VH NUCLEIC ACID* SEQ ID NO: 5410G5-2 HEAVY CHAIN SEQ ID NO: 55 10G5-2 VH SEQ ID NO: 56 10G5-2 HEAVYCHAIN NUCLEIC ACID SEQ ID NO: 57 10G5-2 VH NUCLEIC ACID SEQ ID NO: 5810G5-4,5 HEAVY CHAIN* SEQ ID NO: 59 10G5-4,5 VH* SEQ ID NO: 60 10G5-4,5HEAVY CHAIN NUCLEIC ACID* SEQ ID NO: 61 10G5-4,5 VH NUCLEIC ACID* SEQ IDNO: 62 10G5-6, HEAVY CHAIN* SEQ ID NO: 63 10G5-6, VH* SEQ ID NO: 6410G5-6, HEAVY CHAIN NUCLEIC ACID* SEQ ID NO: 65 10G5-6, VH NUCLEIC ACID*SEQ ID NO: 66 10G5-7,8 HEAVY CHAIN SEQ ID NO: 67 10G5-7,8 VH SEQ ID NO:68 10G5-7,8 HEAVY CHAIN NUCLEIC ACID SEQ ID NO: 69 10G5-7,8 VH NUCLEICACID SEQ ID NO: 70 10G5-1,2,4,6,7 LIGHT CHAIN* SEQ ID NO: 7110G5-1,2,4,6,7 VL* SEQ ID NO: 72 10G5-1,2,4,6,7 LIGHT CHAIN NUCLEICACID* SEQ ID NO: 73 10G5-1,2,4,6,7 VL NUCLEIC ACID* SEQ ID NO: 74 10G5-3LIGHT CHAIN SEQ ID NO: 75 10G5-3 VL SEQ ID NO: 76 10G5-3 LIGHT CHAINNUCLEIC ACID SEQ ID NO: 77 10G5-3 VL NUCLEIC ACID SEQ ID NO: 78 10G5-5,8LIGHT CHAIN* SEQ ID NO: 79 10G5-5,8 VL* SEQ ID NO: 80 10G5-5,8 LIGHTCHAIN NUCLEIC ACID* SEQ ID NO: 81 10G5-5,8 VL NUCLEIC ACID* SEQ ID NO:82 10G5 VH CDR1 SEQ ID NO: 83 10G5 VH CDR2 SEQ ID NO: 84 10G5 VL CDR1SEQ ID NO: 85 10G5 VL CDR2 SEQ ID NO: 86 PFAB3D-10G5H SEQ ID NO: 87PFAB3D-10G5L SEQ ID NO: 92 CONSTANT OF IGG2M4 SEQ ID NO: 93 CONSTANT OFIGG2M4 NUCLEIC ACID SEQ ID NO: 94 10G5-6 HEAVY CHAIN IGG2M4 SEQ ID NO:95 10G5-6 HEAVY CHAIN IGG2M4 NUCLEIC ACID SEQ ID NO: 96 10G5H6 HEAVYCHAIN IGG2M4 SEQ ID NO: 97 IGG1 FC SEQ ID NO: 98 IGG2 FC SEQ ID NO: 99IGG4 FC SEQ ID NO: 100 IGG2M4 FC SEQ ID NO: 101 Mature human IL-13receptor α1 SEQ ID NO: 103 hIL-13Rα1.ECR SEQ ID NO: 104 Maturecynomolgus IL-13Rα1 sequence SEQ ID NO: 105 Mature murine IL-13Rα1sequence SEQ ID NO: 106 VH CDR1 NUCLEIC ACID SEQ ID NO: 107 VH CDR2NUCLEIC ACID SEQ ID NO: 108 VH CDR3 NUCLEIC ACID SEQ ID NO: 109 VL CDR1NUCLEIC ACID SEQ ID NO: 110 VL CDR2 NUCLEIC ACID SEQ ID NO: 111 VL CDR3NUCLEIC ACID SEQ ID NO: 112 VH CDR3 10G5-6 NUCLEIC ACID SEQ ID NO: 113VL CDR3 10G5-6 NUCLEIC ACID SEQ ID NO: 120 Mutant human IL-13 receptorα1 SEQ ID NO: 121 VH CDR3 SEQ ID NO: 122 VL CDR3 SEQ ID NO: 123 VL CDR1NUCLEIC ACID *Note: Where a particular SEQ ID NO: is relevant to morethan one designated antibody, the following format may be utilized as anabbreviation of the different antibodies: Antibody Base Designation-OneAssigned No., Another Assigned No., etc. An example of this is asfollows: 10G5-1,3 refers to both 10G5-1 and 10G5-3.

In specific embodiments, the present invention provides isolatedantibody molecules including a heavy chain variable region of SEQ IDNO:45, equivalents thereof characterized as having one or moreconservative amino acid substitutions, and homologs thereof. Thedisclosed antibodies exhibit at least one of the following functionalproperties: (i) inhibition of IL-13-induced eotaxin release in NHDFcells; (ii) inhibition of IL-4-induced eotaxin release in NHDF cells;(iii) inhibition of IL-13-induced STAT6 phosphorylation in NHDF cells;(iv) inhibition of IL-4-induced STAT6 phosphorylation in NHDF cells; or(v) inhibition of IL-13-stimulated release of TARC in blood or PBMCs. Inspecific embodiments, the present invention provides homologs of thedisclosed antibody molecules characterized as being at least 90%homologous thereto and exhibiting at least one of the above functionalproperties. Specific antibodies provided will compete for binding tohIL-13Rα1 with an antibody as produced by the hybridoma cell linedeposited as ATCC Deposit No. PTA-6933.

In specific embodiments, the present invention provides isolatedantibody molecules including a light chain variable region of SEQ IDNO:49, equivalents thereof characterized as having one or moreconservative amino acid substitutions, and homologs thereof. Thedisclosed antibodies exhibit at least one of the following functionalproperties: (i) inhibition of IL-13-induced eotaxin release in NHDFcells; (ii) inhibition of IL-4-induced eotaxin release in NHDF cells;(iii) inhibition of IL-13-induced STAT6 phosphorylation in NHDF cells;(iv) inhibition of IL-4-induced STAT6 phosphorylation in NHDF cells; and(v) inhibition of IL-13-stimulated release of TARC in blood or PBMCs. Inspecific embodiments, the present invention provides homologs of thedisclosed antibody molecules characterized as being at least 90%homologous thereto and exhibiting at least one of the above functionalproperties. Specific antibodies provided will compete for binding tohIL-13Rα1 with an antibody as produced by the hybridoma cell linedeposited as ATCC Deposit No. PTA-6933.

In specific embodiments, the present invention provides isolatedantibody molecules which comprise a heavy chain variable regioncomprising SEQ ID NO:45 and light chain variable region comprising SEQID NO:49; or equivalent thereof characterized as having one or moreconservative amino acid substitutions. Specific embodiments are saidantibodies which exhibit at least one of the following functionalproperties: (i) inhibition of IL-13-induced eotaxin release in NHDFcells; (ii) inhibition of IL-4-induced eotaxin release in NHDF cells;(iii) inhibition of IL-13-induced STAT6 phosphorylation in NHDF cells;(iv) inhibition of IL-4-induced STAT6 phosphorylation in NHDF cells; and(v) inhibition of IL-13-stimulated release of TARC in blood or PBMCs.

In particular embodiments, the present invention provides isolatedIL-13Rα1 antibody molecules that include the variable heavy CDR3sequence, SEQ ID NO:40, and conservative modifications thereof, whichexhibit at least one of the following functional properties: (i)inhibition of IL-13-induced eotaxin release in NHDF cells; (ii)inhibition of IL-4-induced eotaxin release in NHDF cells; (iii)inhibition of IL-13-induced STAT6 phosphorylation in NHDF cells; (iv)inhibition of IL-4-induced STAT6 phosphorylation in NHDF cells; and (v)inhibition of IL-13-stimulated release of TARC in blood or PBMCs. Heavychain variable region CDR3 sequences particularly embraced by thepresent invention are listed in Table 2.

TABLE 2 SEQ ID NO: V_(H) CDR3 Sequence SEQ ID NO: 1 FPNWGALDQ SEQ ID NO:2 VPNWGSLDT SEQ ID NO: 3 FPNWGSMDA SEQ ID NO: 4 FPNWGSLDH SEQ ID NO: 5MPNWGSFDY SEQ ID NO: 6 MPNWGSFDT SEQ ID NO: 7 MPNWGSLDH SEQ ID NO: 8MPNWGSFDS SEQ ID NO: 9 MPNWGSLDT SEQ ID NO: 10 MPNWGSLDA SEQ ID NO: 11MPNWGSLDN SEQ ID NO: 12 MPNWGALDS SEQ ID NO: 13 MPNWGSFDN SEQ ID NO: 14MPNWGSLDY SEQ ID NO: 15 MPNWGSFDH SEQ ID NO: 16 MPNWGSLDS SEQ ID NO: 17MPNWGSLDG SEQ ID NO: 18 VPNWGSLDN SEQ ID NO: 19CARFPNWGSLDHWGQGTLVTVSSASIKG SEQ ID NO: 20 CARMPNWGSLDHWGQGTLVTVSSASTKGSEQ ID NO: 21 CARMPNWGSFDYWGQGTLVTVSSASIKG SEQ ID NO: 22 VRMPNWGSLDHWSEQ ID NO: 23 VRMPNWGSLDHWGQGTLVTVSSASIKG SEQ ID NO: 24ARMPNWGSLDHWGQGTLVTVSSASIKG SEQ ID NO: 25 FPNWGSFDYWGQGTLVTVSSASIKG SEQID NO: 26 VPNWGSLDA

Specific embodiments provide isolated antibody molecules which include aheavy chain variable region wherein CDR1, CDR2, and/or CDR3 sequencesare SEQ ID NO:82, SEQ ID NO:83 and/or SEQ ID NO:40, respectively; orequivalents thereof characterized as having one or more conservativeamino acid substitutions in any one or more of the CDR sequences.Additional select embodiments provide isolated antibody molecules thatinclude a heavy chain variable region wherein CDR1, CDR2, and/or CDR3sequences are SEQ ID NO:82, SEQ ID NO:102, and/or SEQ ID NO:40,respectively; or equivalents thereof characterized as having one or moreconservative amino acid substitutions in any one or more of the CDRsequences.

In particular embodiments, the present invention provides isolatedIL-13Rα1 antibody molecules which have a variable light CDR3 sequence ofSEQ ID NO:41, and conservative modifications thereof, which exhibit atleast one of the following functional properties: (i) inhibition ofIL-13-induced eotaxin release in NHDF cells; (ii) inhibition ofIL-4-induced eotaxin release in NHDF cells; (iii) inhibition ofIL-13-induced STAT6 phosphorylation in NHDF cells; (iv) inhibition ofIL-4-induced STAT6 phosphorylation in NHDF cells; and (v) inhibition ofIL-13-stimulated release of TARC in blood or PBMCs. Light chain variableregion CDR3 sequences particularly embraced by the present invention arelisted in Table 3.

TABLE 3 SEQ ID NO: V_(L) CDR3 Sequence SEQ ID NO: 27 QRYAT SEQ ID NO: 28QRYST SEQ ID NO: 29 QMYST SEQ ID NO: 30 QQVGT SEQ ID NO: 31 QVYST SEQ IDNO: 32 QQYST SEQ ID NO: 33 QSYST SEQ ID NO: 34 QQYAT SEQ ID NO: 35 QQYSSSEQ ID NO: 36 QTYST SEQ ID NO: 37 QQYGS SEQ ID NO: 38 QQYAS SEQ ID NO:39 QQYEA

Specific embodiments provide isolated antibody molecules which include alight chain variable region wherein CDR1, CDR2, and/or CDR3 sequencesare set forth in SEQ ID NO:84, SEQ ID NO:85, and/or SEQ ID NO:41,respectively; or an equivalent thereof characterized as having one ormore conservative amino acid substitutions in any one or more of the CDRsequences.

In particular embodiments, the present invention provides isolatedIL-13Rα1 antibody molecules which include heavy chain variable regionCDR3 sequence and light chain variable region CDR3 sequence of SEQ IDNOs:40 and 41, respectively, or conservative modifications thereof inany one or more of the CDR3 sequences, that exhibit at least one of thefollowing functional properties: (i) inhibition of IL-13-induced eotaxinrelease in NHDF cells; (ii) inhibition of IL-4-induced eotaxin releasein NHDF cells; (iii) inhibition of IL-13-induced STAT6 phosphorylationin NHDF cells; (iv) inhibition of IL-4-induced STAT6 phosphorylation inNHDF cells; and (v) inhibition of IL-13-stimulated release of TARC inblood or PBMCs.

Specific embodiments provide isolated IL-13Rα1 antibody molecules whichinclude heavy chain variable region CDR1, CDR2, and CDR3 sequences andlight chain variable region CDR1, CDR2, and CDR3 sequences of SEQ IDNOs:82, 83, 40, 84, 85 and 41, respectively; and equivalents thereofcharacterized as having one or more conservative amino acidsubstitutions in any one or more of the CDR sequences.

Conservative amino acid substitutions, as one of ordinary skill in theart will appreciate, are substitutions that replace an amino acidresidue with one imparting similar or better (for the intended purpose)functional and/or chemical characteristics. For example, conservativeamino acid substitutions are often ones in which the amino acid residueis replaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchmodifications do not significantly reduce or alter the binding orfunctional inhibition characteristics of the antibody containing theamino acid sequence but may improve such properties. The purpose formaking a substitution is not significant and can include, but is by nomeans limited to, replacing a residue with one better able to maintainor enhance the structure of the molecule, the charge or hydrophobicityof the molecule, or the size of the molecule. For instance, one maydesire simply to substitute a less desired residue with one of the samepolarity or charge. Such modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. One specific means by which those of skill inthe art accomplish conservative amino acid substitutions is alaninescanning mutagenesis as discussed in, for example, MacLennan et al.,1998 Acta Physiol. Scand. Suppl. 643:55-67, and Sasaki et al., 1998 Adv.Biophys. 35:1-24. The altered antibody molecules are then tested forretained or better function using functional assays available in the artor described herein. Antibody molecules possessing one or more suchconservative amino acid substitutions which retain the ability toselectively bind to hIL-13Rα1 and antagonize IL-13Rα1 functioning at alevel the same or better than the molecule not possessing such aminoacid alterations are referred to herein as “functional equivalents” ofthe disclosed antibodies and form specific embodiments of the presentinvention.

In another aspect, the present invention provides antibody moleculeswhich include heavy and/or light chain variable regions comprising aminoacid sequences that are homologous to the corresponding amino acidsequences of the disclosed antibodies, wherein the antibody moleculesexhibit an equilibrium dissociation constant (K_(D)) of less than 5 nMwith human interleukin 13 receptor α1 (hIL-13Rα1) and antagonizehIL-13Rα1-mediated activity. Specific embodiments are antibody moleculeswhich include heavy and/or light chain variable regions which are atleast 90% homologous to disclosed heavy and/or light chain variableregions, respectively, that exhibit at least one of the followingfunctional properties: (i) inhibition of IL-13-induced eotaxin releasein NHDF cells; (ii) inhibition of IL-4-induced eotaxin release in NHDFcells; (iii) inhibition of IL-13-induced STAT6 phosphorylation in NHDFcells; (iv) inhibition of IL-4-induced STAT6 phosphorylation in NHDFcells; or (v) inhibition of IL-13-stimulated release of TARC in blood orPBMCs. Other embodiments of the present invention are antibody moleculeswhich include heavy and/or light chain variable regions which are atleast 90% homologous to disclosed heavy and/or light chain variableregions, respectively, that compete for binding to hIL-13Rα1 with anantibody as produced by the hybridoma cell line deposited as ATCCDeposit No. PTA-6933. Reference to “at least 90% homologous” in variableregions includes at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and100% homologous sequences.

Antibodies with amino acid sequences homologous to the amino acidsequences of the specific antibody molecules described herein aretypically produced to improve one or more of the properties of theantibody without changing its specificity for IL-13Rα1. One method ofobtaining such sequences, which is not the only method available to theskilled artisan, is to mutate sequence encoding heavy and/or light chainvariable regions disclosed herein by site-directed or randommutagenesis, express an antibody molecule comprising the mutatedvariable region(s), and test the encoded antibody molecule for retainedfunction using the functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and length of each gap, which need to be introduced foroptimal alignment of the two sequences. The comparison of sequences anddetermination of percent identity between sequences can be determinedusing methods generally known to those in the art and can beaccomplished using a mathematical algorithm. For example, the percentidentity between amino acid sequences and/or nucleotide sequences can bedetermined using the algorithm of Meyers and Miller, 1988 Comput. Appl.Biosci. 4:11-17, which has been incorporated into the ALIGN program(version 2.0). In addition, the percent identity between amino acidsequences or nucleotide sequences can be determined using the GAPprogram in the GCG software package available online from Accelrys,using its default parameters.

Specific antibodies of the present invention inhibit the binding ofIL-13 to hIL-13Rα1. Specific antibodies of the present invention competefor binding to hIL-13Rα1 with any of the antibodies disclosed hereinand, particularly, 10G5. Such competing antibodies can be identifiedbased on their ability to cross-compete (e.g., to competitively inhibitthe binding of, in a statistically significant manner) with 10G5 or itsderivatives disclosed herein in standard IL-13Rα1 binding assays. Theability of a test antibody to inhibit the binding of 10G5 or derivativeto human IL-13Rα1 demonstrates that the test antibody can compete withthat antibody for binding to human IL-13Rα1. Such an antibody may,according to non-limiting theory, bind to the same or a related (e.g., astructurally similar or spatially proximal) epitope on human IL-13Rα1 asthe antibody with which it competes. Antibodies that compete for bindingwith 10G5 or its disclosed derivatives may then be assessed for havingat least one of the following functional properties: (i) inhibition ofIL-13-induced eotaxin release in NHDF cells; (ii) inhibition ofIL-4-induced eotaxin release in NHDF cells; (iii) inhibition ofIL-13-induced STAT6 phosphorylation in NHDF cells; (iv) inhibition ofIL-4-induced STAT6 phosphorylation in NHDF cells; and (v) inhibition ofIL-13-stimulated release of TARC in blood or PBMCs. The antibodies canalso then be assessed for binding sensitivity at the Phe233Ala residueof human IL-13Rα1 described herein.

In particular embodiments, the present invention provides isolatedantibody molecules that antagonize IL-13Rα1 function (IL-13Rα1-mediatedactivity) and which exhibit an equilibrium dissociation constant (K_(D))with hIL-13Rα1 which is less than 200 pM, and preferably less than 100pM, as determined by surface plasmon resonance technologies readilyavailable and understood by those of skill in the art, including but notlimited to, BIACORE™ (Upsala, Sweden) and KINEXA® (Sapidyne Instruments,Boise, Id.) or suitable equivalent thereof. In specific embodiments, theisolated antibody molecules exhibit the above K_(D) as well as one ofthe following functional properties: (i) inhibition of IL-13-inducedeotaxin release in NHDF cells; (ii) inhibition of IL-4-induced eotaxinrelease in NHDF cells; (iii) inhibition of IL-13-induced STAT6phosphorylation in NHDF cells; (iv) inhibition of IL-4-induced STAT6phosphorylation in NHDF cells; and (v) inhibition of IL-13-stimulatedrelease of TARC in blood or PBMCs.

In specific embodiments, the present invention provides isolatedantibody molecules which exhibit the above K_(D), antagonizeIL-13Rα1-mediated activity, and comprise a heavy chain variable regionwith a complementarity determining region 3 (CDR3) domain as set forthin SEQ ID NO:5 or an equivalent thereof characterized as havingconservative amino acid substitutions at amino acid positions 1, 7,and/or 9 therein. In specific embodiments, the present inventionprovides isolated antibody molecules which exhibit the above K_(D) andheavy chain variable region and further include a light chain variableregion with a CDR3 domain as set forth in SEQ ID NO:38 or an equivalentthereof characterized as having conservative amino acid substitutions atamino acid positions 2, 4, and/or 5 therein. In specific embodiments,such conservative amino acid substitutions encompass the following:

in SEQ ID NO:5, position 1, a substitution selected from the groupconsisting of: a F, M, Q, L and V;

in SEQ ID NO:5, position 7, a substitution selected from the groupconsisting of: a F, L, A and M;

in SEQ ID NO:5, position 9, a substitution selected from the groupconsisting of: a Y, Q, K, R, W and H;

in SEQ ID NO:38, position 2, a substitution selected from the groupconsisting of: Q, R, M, S and T;

in SEQ ID NO:38, position 4, a substitution selected from the groupconsisting of: E, A, G and S; and/or

in SEQ ID NO:38, position 5, a substitution selected from the groupconsisting of: T, A and S.

Accordingly, one embodiment of the present invention embraces anantibody molecule with a heavy chain variable region CDR3 having thesequence Xaa₁-Pro-Asn-Trp-Gly-Xaa₂-Xaa₃-Asp-Xaa₄ (SEQ ID NO:121),wherein Xaa₁ is Phe, Met, Gln, Leu or Val; Xaa₂ is Ser or Ala; Xaa₃ isPhe, Leu, Ala or Met; and Xaa₄ is Tyr, Gln, Lys, Arg, Trp, His, Ala,Thr, Ser, Asn or Gly. Another embodiment embraces an antibody moleculewith a light chain variable region CDR3 having the sequenceGln-Xaa₁-Xaa₂-Xaa₂-Xaa₄ (SEQ ID NO:122), wherein Xaa₁ is Gln, Arg, Met,Ser, Thr or Val; Xaa₂ is Tyr or Val; Xaa₃ is Glu, Ala, Gly or Ser; andXaa₄ is Thr, Ala or Ser.

One aspect of the present invention is an isolated antibody or antigenbinding fragment that includes:

a heavy chain variable region with a CDR3 domain having a sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, andSEQ ID NO:26; or a heavy chain variable region with a sequence includinga CDR3 domain, said sequence selected from the group consisting of: SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, and SEQ ID NO:25; and/or

a light chain variable region with a CDR3 domain having a sequenceselected from the group consisting of: SEQ ID NO:27, SEQ ID NO:28, SEQID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ IDNO:39;

wherein specific embodiments thereof exhibit a K_(D) with hIL-13Rα1which is less than 200 pM.

Specific embodiments have a heavy chain including a sequence selectedfrom the group consisting of: SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58,SEQ ID NO:62, and SEQ ID NO:66. Specific embodiments have a heavy chainvariable domain including a sequence selected from the group consistingof: SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, and SEQ IDNO:67. Specific embodiments have a light chain having a sequenceselected from the group consisting of: SEQ ID NO:70, SEQ ID NO:74, andSEQ ID NO:78. Specific embodiments have a light chain variable domainhaving a sequence selected from the group consisting of: SEQ ID NO:71,SEQ ID NO:75, and SEQ ID NO:79.

Another aspect of the present invention is an isolated antibody orantigen binding fragment in accordance with the present disclosure thatpossesses the following:

a light chain variable region including a CDR2 domain with the sequenceset forth in SEQ ID NO:85;

a heavy chain variable region including a CDR2 domain with the sequenceset forth in SEQ ID NO:83;

a light chain variable region including a CDR1 domain with the sequenceset forth in SEQ ID NO:84; and/or

a heavy chain variable region including a CDR1 domain with the sequenceset forth in SEQ ID NO:82.

Accordingly, specific embodiments of the present invention provideantibody molecules specific for human IL-13 receptor which include anantigen binding region having heavy and light chain variable regions,and a set of CDRs (CDR1, CDR2, and CDR3) as described herein. Thepresent invention also provides compositions including one and/or bothof the following components (1) a heavy chain variable region having aset of CDRs, and (2) a light chain variable region having a set of CDRs.

In one aspect, the present invention provides isolated antibodymolecules for human IL-13Rα1 which have therein at least one light chainvariable domain and at least one heavy chain variable domain (V_(L) andV_(H), respectively).

In specific embodiments, an antibody molecule having a heavy chainvariable chain region in accordance with the present description isexpressed with a light chain variable region with CDR3 sequence as setforth in SEQ ID NO:41. In other embodiments, an antibody molecule havinga light chain variable region in accordance with the present descriptionis expressed with a heavy chain variable sequence with CDR3 sequence asset forth in SEQ ID NO:40. In specific embodiments, light and heavychains of the formulas described above are used in combination. Specificembodiments of the present invention provide antibody molecules thatfurther include the following:

a light chain variable region with a CDR2 domain as set forth in SEQ IDNO:85;

a heavy chain variable region with a CDR2 domain as set forth in SEQ IDNO:83;

a light chain variable region with a CDR1 domain as set forth in SEQ IDNO:84; and/or

a heavy chain variable region with a CDR1 domain as set forth in SEQ IDNO:82, or suitable equivalents or derivatives thereof, including saiddomains containing conservative amino acid substitutions as describedabove.

Any antibody molecule including the disclosed heavy and/or light CDRs,variable regions or light or heavy chains, or any combination thereof isencompassed within the present invention including, but not limited to,the following antibody molecules: (1) an isolated antibody moleculehaving a heavy chain variable region having therein CDR3 sequence as setforth in SEQ ID NO:5, and a light chain variable region having thereinCDR3 sequence as set forth in SEQ ID NO:38; (2) an isolated antibodymolecule having a heavy chain variable region having therein CDR3sequence as set forth in SEQ ID NO:5, and a light chain variable regionhaving therein CDR3 sequence as set forth in SEQ ID NO:39; (3) anisolated antibody molecule having a heavy chain variable region havingtherein CDR3 sequence as set forth in SEQ ID NO:5, and a light chainvariable region having therein CDR3 sequence as set forth in SEQ IDNO:41; (4) an isolated antibody molecule having a heavy chain variableregion having therein CDR3 sequence as set forth in SEQ ID NO:22, and alight chain variable region having therein CDR3 sequence as set forth inSEQ ID NO:38; (5) an isolated antibody molecule having a heavy chainvariable region having therein CDR3 sequence as set forth in SEQ IDNO:23, and a light chain variable region having therein CDR3 sequence asset forth in SEQ ID NO:38; and (6) an isolated antibody molecule havinga heavy chain variable region having therein CDR3 sequence as set forthin SEQ ID NO:7, and a light chain variable region having therein CDR3sequence as set forth in SEQ ID NO:38.

In some embodiments, an isolated antibody of the present invention has alight chain variable region including a CDR2 domain with the sequenceset forth in SEQ ID NO:85; a heavy chain variable region including aCDR2 domain with the sequence set forth in SEQ ID NO:83; a light chainvariable region including a CDR1 domain with the sequence set forth inSEQ ID NO:84; and a heavy chain variable region including a CDR1 domainwith the sequence set forth in SEQ ID NO:82.

In other embodiments an isolated antibody molecule of the invention hasa heavy chain variable region with CDR1, CDR2 and CDR3 sequences as setforth in SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:7 respectively, and alight chain variable region including CDR1, CDR2 and CDR3 sequences asset forth in SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:38 respectively.

Further encompassed herein are antibody molecules including (i) a heavychain variable region as set forth in SEQ ID NO:63 and/or a light chainvariable region as set forth in SEQ ID NO:71; (ii) a heavy chain as setforth in SEQ ID NO:62 and a light chain variable region as set forth inSEQ ID NO:71, and (iii) a heavy chain as set forth in SEQ ID NO:94 and alight chain variable region as set forth in SEQ ID NO:71.

Manipulation of monoclonal and other antibodies to produce otherantibodies or chimeric molecules which retain the specificity of theoriginal antibody is well within the realm of one skilled in the art.This can be accomplished, for example, using techniques of recombinantDNA technology. Such techniques may involve the introduction of DNAencoding the immunoglobulin variable region, or one or more of the CDRs,of an antibody to the variable region, constant region, or constantregion plus framework regions, as appropriate, of a differentimmunoglobulin. Such molecules form important aspects of the presentinvention. Specific immunoglobulins, into which the disclosed sequencesmay be inserted or, in the alternative, form the essential part of,include but are not limited to the following antibody molecules whichform particular embodiments of the present invention: a Fab (monovalentfragment with variable light (V_(L)), variable heavy (V_(H)), constantlight (C_(L)) and constant heavy 1 (C_(H1)) domains), a F(ab′)₂(bivalent fragment comprising two Fab fragments linked by a disulfidebridge or alternative at the hinge region), a Fd (V_(H) and C_(H1)domains), a Fv (V_(L) and V_(H) domains), a scFv (a single chain Fvwhere V_(L) and V_(H) are joined by a linker, e.g., a peptide linker,see, e.g., Bird et al., 1988 Science 242:423-426, Huston et al., 1988Proc. Natl. Acad. Sci. USA 85:5879-5883), a bispecific antibody molecule(an antibody molecule comprising an IL-13Rα1-specific antibody orantigen binding fragment as disclosed herein linked to a secondfunctional moiety having a different binding specificity than theantibody, including, without limitation, another peptide or protein suchas an antibody, or receptor ligand), a bispecific single chain Fv dimer(see, e.g., PCT/US92/09965), an isolated CDR3, a minibody (singlechain-C_(H3) fusion that self assembles into a bivalent dimer of about80 kDa), a ‘scAb’ (an antibody fragment containing V_(H) and V_(L) aswell as either C_(L) or C_(H1)), a dAb fragment (V_(H) domain, see,e.g., Ward et al., 1989 Nature 341:544-546, and McCafferty et al., 1990Nature 348:552-554; or V_(L) domain; Holt et al., 2003 Trends inBiotechnology 21:484-489), a diabody (see, e.g., Holliger et al., 1993Proc. Natl. Acad. Sci. USA 90:6444-6448 and WO 94/13804), a triabody, atetrabody, a minibody (a scFv joined to a C_(H3); see, e.g., Hu et al.,1996 Cancer Res. 56:3055-3061), IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD,IgA, IgE or any derivatives thereof, and artificial antibodies basedupon protein scaffolds, including but not limited to fibronectin typeIII polypeptide antibodies (see, e.g., U.S. Pat. No. 6,703,199 and WO02/32925) or cytochrome B; see, e.g., Koide et al., 1998 J. Mol. Biol.284:1141-1151, and Nygren et al., 1997 Current Opinion in StructuralBiology 7:463-469. Certain antibody molecules including, but not limitedto, Fv, scFv, and diabody molecules may be stabilized by incorporatingdisulfide bridges to line the V_(H) and V_(L) domains, see, e.g., Reiteret al., 1996 Nature Biotech. 14:1239-1245. Bispecific antibodies may beproduced using conventional technologies (see, e.g., Holliger & Winter,1993 Current Opinion Biotechnol. 4:446-449, specific methods of whichinclude production chemically, or from hybrid hybridomas) and othertechnologies including, but not limited to, the BITE™ technology(molecules possessing antigen binding regions of different specificitywith a peptide linker) and knobs-into-holes engineering (see, e.g.,Ridgeway et al., 1996 Protein Eng. 9:616-621). Bispecific diabodies maybe produced in E. coli, and these molecules as well as other antibodymolecules, as one of skill in the art will appreciate, may be selectedusing phage display in the appropriate libraries (see, e.g., WO94/13804).

Variable domains, into which CDRs of interest are inserted, may beobtained from any germ-line or rearranged human variable domain.Variable domains may also be synthetically produced. The CDR regions canbe introduced into the respective variable domains using recombinant DNAtechnology. One means by which this can be achieved is described inMarks et al., 1992 Bio/Technology 10:779-783. Expression and selectionmay be achieved using suitable technologies including, but not limitedto phage display (see, e.g., WO 92/01047, Kay et al., 1996 Phage Displayof Peptides and Proteins: A Laboratory Manual, San Diego: AcademicPress), yeast display, bacterial display, T7 display, and ribosomedisplay (see, e.g., Lowe & Jermutus, 2004 Curr. Pharm. Biotech.517-527). A variable heavy domain may be paired with a variable lightdomain to provide an antigen binding site. In addition, independentregions (e.g., a variable heavy domain alone) may be used to bindantigen. The artisan is well aware, as well, that two domains of an Fvfragment, V_(L) and V_(H), while perhaps coded by separate genes, may bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (scFvs).

Specific embodiments provide the CDR(s) in germline framework regions.Specific embodiments herein provide heavy chain CDR(s) of interest intoVH5-51 (JH4) in place of the relevant CDR(s); as, for example, in SEQ IDNOs:50, 54, 58, 62, and 66. Specific embodiments herein provide thelight chain CDR(s) into Vκ3 A27 (JK1) in place of the relevant CDR(s);as, for example, in SEQ ID NOs:70, 74, and 78.

The present invention encompasses antibody molecules that are human,humanized, deimmunized, chimeric and primatized. The invention alsoencompasses antibodies produced by the process of veneering; see, e.g.,Mark et al., 1994 Handbook of Experimental Pharmacology, vol. 113: Thepharmacology of monoclonal Antibodies, Springer-Verlag, pp. 105-134 andU.S. Pat. No. 6,797,492. “Human” in reference to the disclosed antibodymolecules specifically refers to antibody molecules having variableand/or constant regions derived from human germline immunoglobulinsequences, wherein said sequences may, but need not, be modified/alteredto have certain amino acid substitutions or residues that are notencoded by human germline immunoglobulin sequence. Such mutations can beintroduced by methods including, but not limited to, random orsite-specific mutagenesis in vitro, or by somatic mutation in vivo.Specific examples of mutation techniques discussed in the literature arethat disclosed in Gram et al., 1992 Proc. Natl. Acad. Sci. USA89:3576-3580; Barbas et al., 1994 Proc. Natl. Acad. Sci. USA91:3809-3813, and Schier et al., 1996 J. Mol. Biol. 263:551-567. Theseare only specific examples and do not represent the only availabletechniques. There are a plethora of mutation techniques in thescientific literature which are available to, and widely appreciated by,the skilled artisan. “Humanized” in reference to the disclosed antibodymolecules refers specifically to antibody molecules wherein CDRsequences derived from another mammalian species, such as a mouse, aregrafted onto human framework sequences. “Primatized” in reference to thedisclosed antibody molecules refers to antibody molecules wherein CDRsequences of a non-primate are inserted into primate frameworksequences, see, e.g., WO 93/02108 and WO 99/55369.

Specific antibodies of the present invention are monoclonal antibodiesand, in particular embodiments, are in one of the following antibodyformats: IgD, IgA, IgE, IgM, IgG1, IgG2, IgG3, IgG4 or any derivative ofany of the foregoing. The language “derivatives thereof” or“derivatives” includes, inter alia, (i) antibodies and antibodymolecules with modifications in the framework or CDR regions of one orboth variable regions (i.e., V_(H) and/or V_(L)), (ii) antibodies andantibody molecules with manipulations in the constant regions of theheavy and/or light chains, and (iii) antibodies and antibody moleculesthat contain additional chemical moieties which are not normally a partof the immunoglobulin molecule (e.g., pegylation).

Manipulations of the variable regions can be within one or more of theV_(H) and/or V_(L) CDR regions. Site-directed mutagenesis or randommutagenesis can be performed to introduce the mutation(s) and the effecton antibody functional property of interest can be evaluated using canbe evaluated by available in vitro or in vivo assays including thosedescribed herein.

Antibodies of the present invention also include those in whichmodifications have been made to the framework residues within V_(H)and/or V_(L) to improve one or more properties of the antibody ofinterest. Typically, such framework modifications are made to decreasethe immunogenicity of the antibody. For example, one approach is to“backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. Such“backmutated” antibodies are also intended to be encompassed by theinvention. Another type of framework modification involves mutating oneor more residues within the framework region, or even within one or moreCDR regions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, where present, typically to alterone or more functional properties of the antibody, such as serumhalf-life, complement fixation, Fc receptor binding, and/orantigen-dependent cellular cytotoxicity.

In one embodiment, the hinge region of C_(H1) is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased, to, for example, facilitate assembly of thelight and heavy chains or to increase or decrease the stability of theantibody. This approach is described further in U.S. Pat. No. 5,677,425by Bodmer et al.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. This approach isdescribed in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: Thr252Leu,Thr254Ser, Thr256Phe, as described in U.S. Pat. No. 6,277,375 by Ward.Alternatively, to increase the biological half-life, the antibody can bealtered within the C_(H1) or CL region to contain a salvage receptorbinding epitope taken from two loops of a C_(H2) domain of an Fc regionof an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 byPresta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, see U.S. Pat. Nos.5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are modified to thereby alter the ability of theantibody to fix complement. This approach is described further in WO94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody-dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids; see for example WO00/42072 by Presta. Moreover, the binding sites on human IgG1 for FcγR1,FcγRII, FcγRIII and FcRn have been mapped and variants with improvedbinding have been described (see Shields et al, J. Biol. Chem.276:6591-6604, 2001).

The concept of generating “hybrids” or “combinatorial” IgG formscomprising various antibody isotypes to hone in on desired effectorfunctionality has generally been described; see, e.g., Tao et al., 1991J. Exp. Med. 173:1025-1028. A specific embodiment of the presentinvention encompasses antibody molecules that possess specificmanipulations in the Fc region which have been found to result inreduced binding to FcγR receptors or C1q on the part of the antibody.The present invention, therefore, encompasses antibodies in accordancewith the present description that do not provoke (or provoke to a lesserextent) antibody-dependent cellular cytotoxicity (ADCC),complement-mediated cytotoxicity (CMC), or form immune complexes, whileretaining normal pharmacokinetic (PK) properties. Specific embodimentsof the present invention provide an antibody molecule as defined inaccordance with the present invention which includes, as part of itsimmunoglobulin structure, the sequence set forth in SEQ ID NO:92. FIG.16 illustrates a comparison of IgG2m4 (as described in U.S. PatentPublication No. US20070148167(A1)), which contains SEQ ID NO:92, withthe amino acid sequence of IgG1, IgG2, and IgG4. One specific example ofthe above-described embodiment is an antibody molecule including SEQ IDNO:94 which possesses sequence based off of a 10G5-6 antibody. Anotherspecific example of the above-described embodiment is an antibodymolecule including SEQ ID NO:96 which possesses sequence based off of an10G5H6 antibody.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation.”

Specific antibody molecules may carry a detectable label, or may beconjugated to a toxin (e.g., a cytotoxin), a radioactive isotope, aradionuclide, a liposome, a targeting moiety, a biosensor, a cationictail, or an enzyme (e.g., via a peptidyl bond or linker). Such antibodymolecule compositions form an additional aspect of the presentinvention.

In another aspect, the present invention provides isolated nucleic acidencoding the disclosed antibody molecules. The nucleic acid may bepresent in whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, forexample, using standard techniques, including without limitation,alkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and other suitable methods known in the art. The nucleicacid may include DNA (inclusive of cDNA) and/or RNA. Nucleic acids ofthe present invention can be obtained using standard molecular biologytechniques. For antibodies expressed by hybridomas (e.g., hybridomasprepared from transgenic mice carrying human immunoglobulin genes),cDNAs encoding the light and heavy chains of the antibody made by thehybridoma can be obtained by standard PCR amplification or cDNA cloningtechniques. For antibodies obtained from an immunoglobulin gene library(e.g., using phage display techniques), nucleic acid encoding theantibody can be recovered from the library.

The present invention encompasses isolated nucleic acid encoding thedisclosed variable heavy and/or light chains and select componentsthereof, particularly the disclosed respective CDR3 regions. In specificembodiments hereof, the CDR(s) are provided within antibody frameworkregions. Specific embodiments provide isolated nucleic acid encoding theCDR(s) inserted into the germline framework regions. Specificembodiments herein provide isolated nucleic acid encoding the heavychain CDR(s) inserted into a VH5-51 (JH4) germline in place of thenucleic acid encoding the corresponding CDR(s); as, for example, in SEQID NOs:52, 56, 60, 64 and 68. Specific embodiments herein provideisolated nucleic encoding the light chain CDR(s) inserted into Vκ3 A27(JK1) germline in place of the nucleic acid encoding the correspondingCDR(s); as, for example, in SEQ ID NOs:72, 76 and 80. The isolatednucleic acid encoding the variable regions can be provided within anydesired antibody molecule format including, but not limited to, thefollowing: F(ab′)₂, a Fab, a Fv, a scFv, bispecific antibody molecules(antibody molecules comprising an IL-13Rα1-specific antibody or antigenbinding fragment as disclosed herein linked to a second functionalmoiety having a different binding specificity than the antibody,including, without limitation, another peptide or protein such as anantibody, or receptor ligand), a bispecific single chain Fv dimer, aminibody, a dAb fragment, diabody, triabody or tetrabody, IgG, IgG1,IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE or any derivatives thereof.

Specific embodiments provide isolated a nucleic acid encoding anantibody molecule including a heavy chain having a sequence selectedfrom the group consisting of SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60,SEQ ID NO:64, and SEQ ID NO:68. Specific embodiments provide isolated anucleic acid encoding an antibody molecule including a heavy chainvariable domain having a sequence selected from the group consisting ofSEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:65,and SEQ ID NO:69. Specific embodiments of the present invention providean isolated nucleic acid encoding an antibody molecule including (i)heavy chain CDR1 nucleotide sequence SEQ ID NO:106, (ii) heavy chainCDR2 nucleotide sequence SEQ ID NO:107, and/or (iii) heavy chain CDR3nucleotide sequence SEQ ID NO:108 or SEQ ID NO:112. Specific embodimentsprovide an isolated nucleic acid encoding an antibody molecule includinga light chain having a sequence selected from the group consisting ofSEQ ID NO:72, SEQ ID NO:76, and SEQ ID NO:80. Specific embodimentsprovide an isolated nucleic acid encoding an antibody molecule includinga light chain variable domain having a sequence selected from the groupconsisting of SEQ ID NO:47, SEQ ID NO:73, SEQ ID NO:77, and SEQ IDNO:81. Specific embodiments of the present invention provide an isolatednucleic acid encoding an antibody molecule including (i) light chainCDR1 nucleotide sequence SEQ ID NO:109 or SEQ ID NO: 123, (ii) lightchain CDR2 nucleotide sequence SEQ ID NO:110, and/or (iii) light chainCDR3 nucleotide sequence SEQ ID NO:111 or SEQ ID NO:113. Specificembodiments of the present invention encompass a nucleic acid encodingan antibody molecule that possesses manipulations in the Fc region whichresult in reduced binding to FcγR receptors or C1q on the part of theantibody. One specific embodiment of the present invention is anisolated nucleic acid which includes SEQ ID NO:93. One specific exampleof such embodiment is an antibody molecule including the sequence of SEQID NO:95, or nucleic acid encoding an antigen binding fragment of SEQ IDNO:94. In specific embodiments, synthetic antibody molecules can beproduced by expression from nucleic acid generated from oligonucleotidessynthesized and assembled within suitable expression vectors; see, e.g.,Knappick et al., 2000 J. Mol. Biol. 296:57-86, and Krebs et al., 2001 J.Immunol. Methods 254:67-84.

Also included within the present invention are nucleic acids includingnucleotide sequences which are at least about 90% identical and morepreferably at least about 95% identical to the nucleotide sequencesdescribed herein, and which nucleotide sequences encode antibodies ofthe present invention. Sequence comparison methods to determine identityare known to those skilled in the art and include those discussedearlier. Reference to “at least about 90% identical” includes at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical.

The invention further provides nucleic acids that hybridize to thecomplement of nucleic acid disclosed herein (e.g., the complement ofnucleic acid including (i) heavy chain nucleotide sequence SEQ ID NO:52,SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68 or SEQ ID NO:95,(ii) V_(H) nucleotide sequence SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:57,SEQ ID NO:61, SEQ ID NO:65 or SEQ ID NO:69; (iii) heavy chain CDR1nucleotide sequence SEQ ID NO:106, (iv) heavy chain CDR2 nucleotidesequence SEQ ID NO:107, (v) heavy chain CDR3 nucleotide sequence SEQ IDNO:108 or SEQ ID NO:112, (vi) light chain nucleotide sequence SEQ IDNO:72, SEQ ID NO:76 or SEQ ID NO:0, (vii) V_(L) nucleotide sequence SEQID NO:47, SEQ ID NO:73, SEQ ID NO:77 or SEQ ID NO:81, (viii) light chainCDR1 nucleotide sequence SEQ ID NO:109 or SEQ ID NO: 123, (ix) lightchain CDR2 nucleotide sequence SEQ ID NO:110, or (x) light chain CDR3nucleotide sequence comprising SEQ ID NO:111 or SEQ ID NO:113) underparticular hybridization conditions, which nucleic acids encode antibodymolecules that bind specifically to hIL-13Rα1 and antagonizeIL-13Rα1-mediated activity. Methods for hybridizing nucleic acids arewell-known in the art; see, e.g., Ausubel, Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1989. Asdefined herein, moderately stringent hybridization conditions may use aprewashing solution containing 5× sodium chloride/sodium citrate (SSC),0.5% w/v SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50%v/v formamide, 6×SSC, and a hybridization temperature of 55° C. (orother similar hybridization solutions, such as one containing about 50%v/v formamide, with a hybridization temperature of 42° C.), and washingconditions of 60° C., in 0.5×SSC, 0.1% w/v SDS. A stringenthybridization condition may be at 6×SSC at 45° C., followed by one ormore washes in 0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill inthe art can manipulate the hybridization and/or washing conditions toincrease or decrease the stringency of hybridization such that nucleicacids comprising nucleotide sequences that are at least 65, 70, 75, 80,85, 90, 95, 98, or 99% identical to each other typically remainhybridized to each other. The basic parameters affecting the choice ofhybridization conditions and guidance for devising suitable conditionsare set forth by Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,Chapters 9 and 11, 1989; and Ausubel et al. (eds), Current Protocols inMolecular Biology, John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4,1995, and can be readily determined by those having ordinary skill inthe art based on, for example, the length and/or base composition of theDNA.

The present invention provides isolated antibodies which include a lightand/or heavy chain variable domain that is encoded at least in part by anucleotide sequence that hybridizes under moderately stringentconditions to the complement of a nucleic acid sequence encoding a lightand/or heavy chain variable domain disclosed herein (e.g., selected fromthe group consisting of SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:53, SEQ IDNO:57, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73, SEQ IDNO:77 and SEQ ID NO:81). In another embodiment, the present inventionencompasses isolated antibodies which include a light and/or heavy chainvariable domain that is encoded at least in part by a nucleotidesequence that hybridizes under stringent conditions to the complement ofa nucleic acid sequence comprising a light and/or heavy chain variabledomain disclosed herein.

In another aspect, the present invention provides vectors including saidnucleic acid. Vectors in accordance with the present invention include,but are not limited to, plasmids and other expression constructs (e.g.,phage or phagemid, as appropriate) suitable for the expression of thedesired antibody molecule at the appropriate level for the intendedpurpose; see, e.g., Sambrook & Russell, Molecular Cloning: A LaboratoryManual: 3^(rd) Edition, Cold Spring Harbor Laboratory Press. For mostcloning purposes, DNA vectors may be used. Typical vectors includeplasmids, modified viruses, bacteriophage, cosmids, yeast artificialchromosomes, and other forms of episomal or integrated DNA. It is wellwithin the purview of the skilled artisan to determine an appropriatevector for a particular gene transfer, generation of a recombinant humanantibody, or other use. In specific embodiments, in addition to arecombinant gene, the vector may also contain an origin of replicationfor autonomous replication in a host cell, appropriate regulatorysequences, such as a promoter, a termination sequence, a polyadenylationsequence, an enhancer sequence, a selectable marker, a limited number ofuseful restriction enzyme sites, other sequences as appropriate and thepotential for high copy number. Examples of expression vectors forantibody and antibody fragment production are well-known in the art;see, e.g., Persic et al., 1997 Gene 187:9-18; Boel et al., 2000 J.Immunol. Methods 239:153-166, and Liang et al., 2001 J. Immunol. Methods247:119-130. If desired, nucleic acid encoding an antibody may beintegrated into the host chromosome using techniques well-known in theart; see, e.g., Ausubel, Current Protocols in Molecular Biology, JohnWiley & Sons, 1999, and Marks et al., WO 95/17516. Nucleic acids mayalso be expressed on plasmids maintained episomally or incorporated intoan artificial chromosome; see, e.g., Csonka et al., 2000 J. Cell Science113:3207-3216; Vanderbyl et al., 2002 Molecular Therapy 5:10. Theantibody light chain gene and the antibody heavy chain gene can beinserted into separate vector or, more typically, both genes areinserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype by inserting them into expression vectors already encoding heavychain constant and light chain constant regions of the desired isotypesuch that the V_(H) segment is operatively linked to the C_(H)segment(s) within the vector and the V_(L) segment is operatively linkedto the C_(L) segment within the vector. Additionally or alternatively,the recombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein). Any technique available to the skilledartisan may be employed to introduce the nucleic acid into the hostcell; see, e.g., Morrison, 1985 Science, 229:1202. Methods of subcloningnucleic acid molecules of interest into expression vectors, transformingor transfecting host cells containing the vectors, and methods of makingsubstantially pure protein including the steps of introducing therespective expression vector into a host cell, and cultivating the hostcell under appropriate conditions are well-known. The antibody soproduced may be harvested from the host cells in conventional ways.Techniques suitable for the introduction of nucleic acid into cells ofinterest will depend on the type of cell being used. General techniquesinclude, but are not limited to, calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using viruses appropriate to the cell line of interest(e.g., retrovirus, vaccinia, baculovirus, or bacteriophage).

In another aspect, the present invention provides isolated cell(s)including nucleic acid encoding the disclosed antibody molecules andcomponents thereof as described. A variety of different cell lines canbe used for recombinant production of antibody molecules, including butnot limited to those from prokaryotic organisms (e.g., E. coli,Bacillus, and Streptomyces) and from eukaryotic (e.g., yeast,Baculovirus, and mammalian); see, e.g., Breitling et al., Recombinantantibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag,1999. Plant cells, including transgenic plants, and animal cells,including transgenic animals (other than humans), including the nucleicacid or antibody molecules disclosed herein are also contemplated aspart of the present invention. Suitable mammalian cell lines including,but not limited to, those derived from Chinese Hamster Ovary (CHO)cells, including but not limited to DHFR-CHO cells (described in Urlauband Chasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220) used, forexample, with a DHFR selectable marker (e.g., as described in Kaufmanand Sharp, 1982 Mol. Biol. 159:601-621), NS0 myeloma cells (where a GSexpression system as described in WO 87/04462, WO 89/01036, and EP338,841 may be used), COS cells, SP2 cells, HeLa cells, baby hamsterkidney cells, YB2/0 rat myeloma cells, human embryonic kidney cells,human embryonic retina cells, and others harboring the nucleic acid orantibody molecules disclosed herein form additional embodiments of thepresent invention. Specific embodiments of the present invention mayemploy E. coli; see, e.g., Plückthun, 1991 Bio/Technology 9:545-551, oryeast, such as Pichia, and recombinant derivatives thereof (see, e.g.,Li et al., 2006 Nat. Biotechnol. 24:210-215). Additional specificembodiments of the present invention may employ eukaryotic cells for theproduction of antibody molecules, see, Chadd & Chamow, 2001 CurrentOpinion in Biotechnology 12:188-194, Andersen & Krummen, 2002 CurrentOpinion in Biotechnology 13:117, Larrick & Thomas, 2001 Current Opinionin Biotechnology 12:411-418. Specific embodiments of the presentinvention may employ mammalian cells able to produce antibody moleculeswith proper post-translational modifications. Post-translationalmodifications include, but are by no means limited to, disulfide bondformation and glycosylation. Another type of post-translationalmodification is signal peptide cleavage. Specific embodiments hereinhave the appropriate glycosylation; see, e.g., Yoo et al., 2002 J.Immunol. Methods 261:1-20. Naturally occurring antibodies contain atleast one N-linked carbohydrate attached to a heavy chain. Id. Differenttypes of mammalian host cells can be used to provide for efficientpost-translational modifications. Examples of such host cells includeChinese Hamster Ovary (CHO), HeLa, C6, PC12, and myeloma cells; see, Yooet al., 2002J. Immunol. Methods 261:1-20, and Persic et al., 1997 Gene187:9-18.

In another aspect, the present invention provides isolated cell(s)comprising a polypeptide of the present invention.

In another aspect, the present invention provides a method of making anantibody molecule of the present invention, which involves incubating acell harboring a nucleic acid encoding a heavy and/or light chain(dictated by the desired antibody molecule) with specificity for humanIL-13Rα1 under conditions that allow the expression and assembly of saidheavy and/or light chains into an antibody molecule, and isolating saidantibody molecule from the cell. One example by which to generate thedesired heavy and/or light chain sequence is to first amplify (andmodify) the germline heavy and/or light chain variable sequences usingPCR. Germline sequence for human heavy and/or light variable regions arereadily available to the skilled artisan, see, e.g., the “Vbase” humangermline sequence database, and Kabat, E. A. et al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M.et al., 1992 “The Repertoire of Human Germline VH Sequences Revealsabout Fifty Groups of VH Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 “A Directory ofHuman Germ-line Vκ Segments Reveals a Strong Bias in their Usage” Eur.J. Immunol. 24:827-836. Mutagenesis of the germline sequences may becarried out using standard methods, e.g., PCR-mediated mutagenesis wherethe mutations are incorporated into the PCR primers, or site-directedmutagenesis. If full-length antibodies are desired, sequence isavailable for the human heavy chain constant region genes; see, e.g.,Kabat. E. A. et al., 1991 Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242. Fragments containing these regions may beobtained, for example, by standard PCR amplification. Alternatively, theskilled artisan can avail him/herself of vectors already encoding heavyand/or light chain constant regions.

Available techniques exist to recombinantly produce other antibodymolecules which retain the specificity of an original antibody. Aspecific example of this is where DNA encoding the immunoglobulinvariable region or the CDRs is introduced into the constant regions, orconstant regions and framework regions, of another antibody molecule;see, e.g., EP-184,187, GB 2188638, and EP-239400, and scientificliterature in the area. Cloning and expression of antibody molecules,including chimeric antibodies, are described in the literature; see,e.g., EP 0120694 and EP 0125023, and other scientific literature in thearea.

Additional antibodies in accordance with the present invention can beraised and then screened for the characteristics identified herein usingknown techniques. Basic techniques for the preparation of monoclonalantibodies are described in the literature, see, e.g., Kohler andMilstein (1975, Nature 256:495-497). Fully human monoclonal antibodiesare produced by available methods. These methods include, but are by nomeans limited to, the use of genetically engineered mouse strains whichpossess an immune system whereby the mouse antibody genes have beeninactivated and in turn replaced with a repertoire of functional humanantibody genes, while leaving other components of the mouse immunesystem unchanged. Such genetically engineered mice allow for the naturalin vivo immune response and affinity maturation process which results inhigh affinity, full human monoclonal antibodies. This technology iswell-known in the art and is fully detailed in various publicationsincluding, but not limited to, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; 5,770,249 (assigned to GenPharm International and availablethrough Medarex, under the umbrella of the “UltraMab Human AntibodyDevelopment System”); as well as U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and related family members (assigned to Abgenix,disclosing their XENOMOUSE® technology). See also reviews from Kellermanand Green, 2002 Curr. Opinion in Biotechnology 13:593-597, andKontermann & Stefan, 2001 Antibody Engineering, Springer LaboratoryManuals.

Alternatively, a library of antigen binding fragments in accordance withthe present invention may be brought into contact with IL-13Rα1, andones able to demonstrate binding at the prescribed level, e.g.,exhibiting a K_(D) with the antigen which is less than 200 pM and theability to antagonize IL-13Rα1-mediated activity selected. Techniquesare available to the artisan for the selection of antibody fragmentsfrom libraries using enrichment technologies including, but not limitedto, phage display (e.g., see technology from Cambridge AntibodyTechnology (CAT) disclosed in U.S. Pat. Nos. 5,565,332; 5,733,743;5,871,907; 5,872,215; 5,885,793; 5,962,255; 6,140,471; 6,225,447;6,291,650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081, as well as other U.S. family members and/or applicationswhich rely on priority filing GB 9206318, filed May 24, 1992; see alsoVaughn et al., 1996, Nature Biotechnology 14:309-314), ribosome display(see, e.g., Hanes and Pluckthün, 1997 Proc. Natl. Acad. Sci.94:4937-4942), bacterial display (see, e.g., Georgiou, et al., 1997Nature Biotechnology 15:29-34) and/or yeast display (see, e.g., Kieke,et al., 1997 Protein Engineering 10:1303-1310). A library, for example,can be displayed on the surface of bacteriophage particles, with thenucleic acid encoding the antigen binding fragments expressed anddisplayed on its surface. Nucleic acids may then be isolated frombacteriophage particles exhibiting the desired level of activity and thenucleic acids used in the development of antibody molecules. Individualheavy or light chain clones in accordance with the present invention mayalso be used to screen for complementary heavy or light chains,respectively, capable of interaction therewith to form a molecule of thecombined heavy and light chains; see, e.g., WO 92/01047. Phage displayhas been described in the literature; see, e.g., Kontermann & Stefan,supra, and WO 92/01047.

Monoclonal antibodies (MAbs) may be purified by techniques available toone of skill in the art. Antibody titers of the relevant ascites,hybridoma culture fluids, or test sample of interest can be determinedby various serological or immunological assays which include, but arenot limited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) techniques and radioimmunoassay (RIA)techniques.

In another aspect, the present invention provides a method forantagonizing the activity of IL-13Rα1, which involves contacting a cellexpressing IL-13Rα1 with an antibody molecule disclosed herein underconditions that allow said antibody molecule to bind to IL-13Rα1.Specific embodiments of the present invention include such methodswherein the cell is a human cell.

In another aspect, the present invention provides a method forantagonizing the activity of IL-13Rα1 in a subject exhibiting acondition associated with IL-13Rα1 activity, which involvesadministering to the subject a therapeutically effective amount of anantibody molecule of the present invention. “Antagonizing” herein refersto the act of opposing, counteracting or curtailing one or morefunctions of the target, be that binding, signaling or other. Inhibitionor antagonism of one or more of the IL-13Rα1 functional properties canbe readily determined according to methodologies known to the art aswell as those described herein. It will, furthermore, be understood thatsuch inhibition or antagonism should effectuate a decrease in theparticular activity relative to that seen in the absence of the antibodyor, for example, that seen when a control antibody of irrelevantspecificity is present. Preferably, an antibody molecule in accordancewith the present invention antagonizes IL-13 and/or IL-4-mediatedIL-13Rα1 functioning to the point that there is a decrease of at least10%, of the measured parameter, and more preferably, a decrease of atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the measuredparameter. Such inhibition/antagonism of IL-13Rα1 functioning isparticularly effective in those instances where receptor functioning iscontributing at least in part to a particular phenotype, disease,disorder or condition which is negatively impacting the subject.

Also contemplated are methods of using the disclosed antibody moleculesin the manufacture of a medicament for treatment of an IL-13Rα1-mediateddisease, disorder or condition. Thus, in another aspect, the presentinvention provides a pharmaceutically acceptable composition comprisingan antibody molecule of the invention and a pharmaceutically acceptablecarrier, excipient, diluent, stabilizer, buffer, or alternative designedto facilitate administration of the antibody molecule in the desiredformat and amount to the treated individual. Antibody moleculesdisclosed herein can be used in a method of treatment or diagnosis of aparticular individual (human or primate). The method of treatment can beprophylactic or therapeutic in nature. Methods of treatment inaccordance with the present invention include administering to anindividual a therapeutically (or prophylactically) effective amount ofan antibody molecule of the present invention. “Therapeuticallyeffective” or “prophylactically effective” amount refers to the amountnecessary at the intended dosage to achieve the desiredtherapeutic/prophylactic effect for the period of time desired. Thedesired effect may be, for example, amelioration of at least one symptomassociated with the treated condition. These amounts will vary, as theskilled artisan will appreciate, according to various factors, includingbut not limited to the disease state, age, sex and weight of theindividual, and the ability of the antibody molecule to elicit thedesired effect in the individual. The response may be documented by invitro assay, in vivo non-human animal studies, and/or further supportedfrom clinical trials. The antibody-based pharmaceutical composition ofthe present invention may be formulated by any number of strategiesknown in the art, see, e.g., McGoff and Scher, 2000 Solution Formulationof Proteins/Peptides, In: McNally, E. J., ed. Protein Formulation andDelivery, New York, N.Y.: Marcel Dekker; pp. 139-158; Akers &Defilippis, 2000, Peptides and Proteins as Parenteral Solutions, In:Pharmaceutical Formulation Development of Peptides and Proteins,Philadelphia, Pa.: Taylor and Francis; pp. 145-177; Akers et al., 2002,Pharm. Biotechnol. 14:47-127. A pharmaceutically acceptable compositionsuitable for patient administration will contain an effective amount ofthe antibody molecule in a formulation which both retains biologicalactivity while also promoting maximal stability during storage within anacceptable temperature range.

The antibody-based pharmaceutically acceptable composition may be inliquid or solid form. Any technique for production of liquid or solidformulations may be utilized. Such techniques are well within the realmof the abilities of the skilled artisan. Solid formulations may beproduced by any available method including, but not limited to,lyophilization, spray drying, or drying by supercritical fluidtechnology. Solid formulations for oral administration may be in anyform rendering the antibody molecule accessible to the patient in theprescribed amount and within the prescribed period of time. The oralformulation can take the form of a number of solid formulationsincluding, but not limited to, a tablet, capsule, or powder. Solidformulations may alternatively be lyophilized and brought into solutionprior to administration for either single or multiple dosing. Antibodycompositions should generally be formulated within a biologicallyrelevant pH range and may be buffered to maintain a proper pH rangeduring storage. Both liquid and solid formulations generally requirestorage at lower temperatures (e.g., 2-8° C.) in order to retainstability for longer periods. Formulated antibody compositions,especially liquid formulations, may contain a bacteriostat to prevent orminimize proteolysis during storage, including but not limited toeffective concentrations (e.g., ≦1% w/v) of benzyl alcohol, phenol,m-cresol, chlorobutanol, methylparaben, and/or propylparaben. Abacteriostat may be contraindicated for some patients. Therefore, alyophilized formulation may be reconstituted in a solution eithercontaining or not containing such a component. Additional components maybe added to either a buffered liquid or solid antibody formulation,including but not limited to sugars as a cryoprotectant (including butnot limited to polyhydroxy hydrocarbons such as sorbitol, mannitol,glycerol, and dulcitol and/or disaccharides such as sucrose, lactose,maltose, or trehalose) and, in some instances, a relevant salt(including but not limited to NaCl, KCl, or LiCl). Such antibodyformulations, especially liquid formulations slated for long termstorage, will rely on a useful range of total osmolarity to both promotelong term stability at temperatures of, for example, 2-8° C. or higher,while also making the formulation useful for parenteral injection. Asappropriate, preservatives, stabilizers, buffers, antioxidants and/orother additives may be included. The formulations may contain a divalentcation (including but not limited to MgCl₂, CaCl₂, and MnCl₂); and/or anon-ionic surfactant (including but not limited to Polysorbate-80 (TWEEN80™), Polysorbate-60 (TWEEN 60™), Polysorbate-40 (TWEEN 40™), andPolysorbate-20 (TWEEN 20™), polyoxyethylene alkyl ethers, including butnot limited to BRIJ 58™, BRIJ 35™, as well as others such as TRITONX-100™, TRITON X-114™, NP40™, Span 85 and the PLURONIC® series ofnon-ionic surfactants (e.g., PLURONIC® 121). Any combination of suchcomponents form specific embodiments of the present invention.

Pharmaceutical compositions in liquid format may include a liquidcarrier, e.g., water, petroleum, animal oil, vegetable oil, mineral oil,or synthetic oil. The liquid format may also include physiologicalsaline solution, dextrose or other saccharide solution or glycols, suchas ethylene glycol, propylene glycol or polyethylene glycol.

The pharmaceutical composition may be in the form of a parenterallyacceptable aqueous solution that is pyrogen-free with suitable pH,tonicity, and stability. Pharmaceutical compositions may be formulatedfor administration after dilution in isotonic vehicles, for example,Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer'sInjection.

Dosing of antibody therapeutics is well within the realm of the skilledartisan, see, e.g., Lederman et al., 1991 Int. J. Cancer 47:659-664;Bagshawe et al., 1991 Antibody, Immunoconjugates andRadiopharmaceuticals 4:915-922, and will vary based on a number offactors including but not limited to the antibody molecule utilized, thepatient being treated, the condition of the patient, the area beingtreated, the route of administration, and the treatment desired. Aphysician or veterinarian of ordinary skill can readily determine andprescribe the effective therapeutic amount of the antibody. Dosageranges may be from about 0.01 to 100 mg/kg, and more usually 0.05 to 25mg/kg, of the host body weight. For example, dosages can be 0.3 mg/kgbody weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg bodyweight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Forpurposes of illustration, and not limitation, in specific embodiments, adose of 5 mg to 2.0 g may be utilized to deliver the antibody moleculesystemically. Optimal precision in achieving concentrations of antibodywithin a range that yields efficacy without toxicity requires a regimenbased on the kinetics of the drug's availability to the target site(s).This involves a consideration of the distribution, equilibrium, andelimination of the antibody molecule. Antibodies described herein may beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents may be desirable. It will bepossible to present a therapeutic dosing regime for the antibodymolecules of the present invention in conjunction with alternativetreatment regimes. Individuals (subjects) capable of treatment includeprimates, human and non-human, and include any non-human mammal orvertebrate of commercial or domestic veterinary importance.

The antibody molecule could be administered to an individual by anyroute of administration appreciated in the art, including but notlimited to oral administration, administration by injection (specificembodiments of which include intravenous, subcutaneous, intraperitonealor intramuscular injection), administration by inhalation, intranasal,or topical administration, either alone or in combination with otheragents designed to assist in the treatment of the individual. The routeof administration should be determined based on a number ofconsiderations appreciated by the skilled artisan including, but notlimited to, the desired physiochemical characteristics of the treatment.Treatment may be provided on a daily, weekly, biweekly, or monthlybasis, or any other regimen that delivers the appropriate amount ofantibody molecule to the individual at the prescribed times such thatthe desired treatment is effected and maintained. The formulations maybe administered in a single dose or in more than one dose at separatetimes.

In particular embodiments, the condition treated is selected from thegroup consisting of asthma, allergy, allergic rhinitis, chronicsinusitis, hay fever, atopic dermatitis, chronic obstructive pulmonarydisease (COPD), pulmonary fibrosis, esophageal eosinophilia, psoriasis,psoriatic arthritis, fibrosis, scleroderma, inflammatory bowel disease(particularly, ulcerative colitis), anaphylaxis, and cancer(particularly, Hodgkin's lymphoma, glioma, and renal carcinoma), andgeneral Th2-mediated disorders/conditions.

The present invention further provides for the administration of thedisclosed anti-hIL-13Rα1 antibody molecules for purposes of genetherapy. In such a method, the cells of a subject would be transformedwith nucleic acid encoding the antibody molecules of the invention.Subjects comprising the nucleic acids will then produce the antibodymolecules endogenously. Previously, Alvarez, et al, Clinical CancerResearch 6:3081-3087, 2000, introduced single-chain anti-ErbB2antibodies to subjects using a gene therapy approach. The methodsdisclosed by Alvarez, et al, may be easily adapted for the introductionof nucleic acids encoding an anti-hIL-13Rα1 antibody of the invention toa subject.

Nucleic acids encoding any polypeptide or antibody molecule of theinvention may be introduced to a subject. In specific embodiments, theantibody molecule is a human, single-chain antibody.

The nucleic acids may be introduced to the cells of a subject by anymeans known in the art. In specific embodiments, the nucleic acids areintroduced as part of a viral vector. Examples of particular virusesfrom which the vectors may be derived include lentiviruses, herpesviruses, adenoviruses, adeno-associated viruses, vaccinia virus,baculovirus, alphavirus, influenza virus, and other recombinant viruseswith desirable cellular tropism.

Various companies produce viral vectors commercially, including, but byno means limited to, AVIGEN, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), CLONTECH (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), GENVEC(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Methods for constructing and using viral vectors are known in the art(see, e.g., Miller, et al, BioTechniques 7:980-990, 1992). In specificembodiments, the viral vectors are replication defective, that is, theyare unable to replicate autonomously, and thus are not infectious, inthe target cell. The replication defective virus may be a minimal virus,i.e., it retains only the sequences of its genome which are necessaryfor encapsidating the genome to produce viral particles. Defectiveviruses which entirely or almost entirely lack viral genes may be usedas well. Use of defective viral vectors allows for administration tocells in a specific, localized area, without concern that the vector caninfect other cells. Thus, a specific tissue can be specificallytargeted.

Examples of vectors comprising attenuated or defective DNA virussequences include, but are not limited to, a defective herpes virusvector (Kanno et al, Cancer Gen. Ther. 6:147-154, 1999; Kaplitt et al,J. Neurosci. Meth. 71:125-132, 1997 and Kaplitt et al, J. Neuro Onc.19:137-147, 1994).

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Attenuated adenovirus vectors, such as the vector described byStrafford-Perricaudet et al, J. Clin. Invest. 90:626-630, 1992 aredesirable in some instances. Various replication defective adenovirusand minimum adenovirus vectors have been described (see, e.g.,WO94/26914, WO94/28938, WO94/28152, WO94/12649, WO95/02697 andWO96/22378). The replication defective recombinant adenovirusesaccording to the invention can be prepared by any technique known to aperson skilled in the art (Levrero et al, Gene 101:195, 1991; EP 185573;Graham, EMBO J. 3:2917, 1984; Graham et al, J. Gen. Virol. 36:59, 1977).

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize which can integrate, in a stable and site-specific manner, into thegenome of the cells which they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (see Daly, etal, Gene Ther. 8:1343-1346, 2001, Larson et al, Adv. Exp. Med. Bio.489:45-57, 2001; WO 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368and 5,139,941 and EP 488528B1).

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764,4,980,289, and 5,124,263; Mann et al, Cell 33:153, 1983; Markowitz etal, J. Virol., 62:1120, 1988; EP 453242 and EP178220. The retrovirusesare integrating viruses which infect dividing cells.

Lentiviral vectors can be used as agents for the direct delivery andsustained expression of nucleic acids encoding an antibody molecule ofthe invention in several tissue types, including brain, retina, muscle,liver and blood. The vectors can efficiently transduce dividing andnondividing cells in these tissues, and maintain long-term expression ofthe antibody molecule. For a review, see Zufferey et al, J. Virol.72:9873-80, 1998 and Kafri et al, Curr. Opin. Mol. Ther. 3:316-326,2001. Lentiviral packaging cell lines are available and known generallyin the art. They facilitate the production of high-titer lentivirusvectors for gene therapy. An example is a tetracycline-inducible VSV-Gpseudotyped lentivirus packaging cell line which can generate virusparticles at titers greater than 10⁶ IU/ml for at least 3 to 4 days; seeKafri et al, J. Virol. 73:576-584, 1999. The vector produced by theinducible cell line can be concentrated as needed for efficientlytransducing nondividing cells in vitro and in vivo.

Sindbis virus is a member of the alphavirus genus and has been studiedextensively since its discovery in various parts of the world beginningin 1953. Gene transduction based on alphavirus, particularly Sindbisvirus, has been well-studied in vitro (see Straus et al, Microbiol.Rev., 58:491-562, 1994; Bredenbeek et al, J. Virol., 67:6439-6446, 1993;Ijima et al, Int. J. Cancer 80:110-118, 1999 and Sawai et al, Biochim.Biophyr. Res. Comm. 248:315-323, 1998. Many properties of alphavirusvectors make them a desirable alternative to other virus-derived vectorsystems being developed, including rapid engineering of expressionconstructs, production of high-titered stocks of infectious particles,infection of nondividing cells, and high levels of expression (Strausset al, 1994 supra). Use of Sindbis virus for gene therapy has beendescribed. (Wahlfors et al, Gene. Ther. 7:472-480, 2000 and Lundstrom,J. Recep. Sig. Transduct. Res. 19(1-4):673-686, 1999.

In another embodiment, a vector can be introduced to cells bylipofection or with other transfection facilitating agents (peptides,polymers, etc.). Synthetic cationic lipids can be used to prepareliposomes for in vivo and in vitro transfection of a gene encoding amarker (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987 andWang et al, Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987). Useful lipidcompounds and compositions for transfer of nucleic acids are describedin WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., electroporation,microinjection, cell fusion, DEAE-dextran, calcium phosphateprecipitation, use of a gene gun, or use of a DNA vector transporter(see, e.g., Wilson, et al, J. Biol. Chem. 267:963-967, 1992; Williams etal, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Receptor-mediatedDNA delivery approaches can also be used (Wu et al, J. Biol. Chem.263:14621-14624, 1988). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclosedelivery of exogenous DNA sequences, free of transfection facilitatingagents, in a mammal. Recently, a relatively low voltage, high efficiencyin vivo DNA transfer technique, termed electrotransfer, has beendescribed (Vilquin et al, Gene Ther. 8:1097, 2001; Payen et al, Exp.Hematol. 29:295-300, 2001; Mir, Bioelectrochemistry 53:1-10, 2001; WO99/01157, WO 99/01158 and WO 99/01175).

Pharmaceutical compositions suitable for such gene therapy approachesand comprising nucleic acids encoding an anti-hIL-13Rα1 antibodymolecule of the present invention are included within the scope of thepresent invention.

In another aspect, the present invention provides a method foridentifying, isolating, quantifying or antagonizing IL-13Rα1 in a sampleof interest using an antibody molecule of the present invention. Theantibody molecules may be utilized as a research tool in immunochemicalassays, such as western blots, ELISAs, radioimmunoassay,immunohistochemical assays, immunoprecipitations, or otherimmunochemical assays known in the art (see, e.g., ImmunologicalTechniques Laboratory Manual, ed. Goers, J. 1993, Academic Press) orvarious purification protocols. The antibody molecules may have a labelto facilitate ready identification or measurement of the activitiesassociated therewith. One skilled in the art is readily familiar withthe various types of detectable labels (e.g., enzymes, dyes, or othersuitable molecules which are either readily detectable or cause someactivity/result that is readily detectable) useful in the aboveprotocols.

Additional aspects of the present invention are kits including theantibody molecules or pharmaceutical compositions disclosed herein andinstructions for use. Kits typically but need not include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

Related to yet another aspect of the present invention, a criticalcontact point between the antibodies disclosed herein and the hIL-13Rα1receptor was identified. This critical contact point was identified byintroducing a specific mutation that impacted the binding of thereceptor by the antibody. More specifically, it was found thatsubstitution of the phenylalanine residue at position 233 of SEQ IDNO:101 with an alanine residue results in a loss of binding between theantibody and the mutant receptor compared with the binding between theantibody and wild type receptor. This is a very useful finding incharacterizing 10G5 and its derivatives. Peptides exploiting the regionsurrounding amino acid 233 will be useful in the generation ofadditional monoclonal antibodies with similar specificity. Accordingly,the present invention encompasses isolated peptides including a hIL-13Rsequence with a Phe233Ala mutation (i.e. SEQ ID NO:120), whether they befull-length or fragments thereof (e.g., 5 to 350 amino acid residuefragments) including the mutation. Additionally, the present inventionalso encompasses the use of said peptides in an assay to evaluate 10G5or 10G5 derivatives, identify antibodies with specificity for a similarepitope or, alternatively, produce antibodies with similar specificity.In specific embodiments the present invention, therefore, provides amethod of screening for antibody molecules that target the same regionon hIL-13Rα1 as the antibody produced by the hybridoma cell linedeposited as ATCC Deposit No. PTA-6933, which involves (a) analyzingbinding of an antibody molecule of interest to (i) an isolated hIL-13Rα1polypeptide of SEQ ID NO:101 or fragment thereof including the aminoacid corresponding to residue 233 of SEQ ID NO:101; and (ii) an isolatedpolypeptide or fragment of an isolated hIL-13Rα1 polypeptide or fragmentthereof wherein the amino acid corresponding to residue 233 of SEQ IDNO:101 is an alanine rather than a phenylalanine; (b) identifying thoseantibodies that bind to the isolated polypeptide or fragment of step(a)(i) but exhibit significantly reduced binding to the isolatedpolypeptide or fragment of step (a) (ii); and (c) recovering theantibodies identified in step (b). A significant decrease is typicallymore than a 10-fold reduction in binding between an antibody and themutant receptor compared with the binding between the antibody and wildtype receptor. Polypeptides that are useful in the method are thosefragments of SEQ ID NO:101 including the amino acid corresponding toresidue 233 of SEQ ID NO:101 that bind to the antibody produced by thehybridoma cell line deposited as ATCC Deposit No. PTA-6933, and thecorresponding fragments wherein the amino acid corresponding to residue233 of SEQ ID NO:101 is an alanine rather than a phenylalanine. Forexample, the extracellular region of human IL-13Rα1 (amino acids number1 to 317 of SEQ ID NO:101) may be used along with its correspondingPhe233Ala mutant.

Another aspect of the invention includes an isolated antibody that (i)competes with the antibody produced by the hybridoma cell line depositedas ATCC Deposit No. PTA-6933 for binding to hIL-13Rα1; (ii) inhibitsIL-13 signaling; and (iii) wherein a substitution in a hIL-13Rα1 peptideof the phenylalanine residue that corresponds to position 233 of SEQ IDNO:101 with an alanine residue leads to a loss of binding between saidantibody and the resultant mutant hIL-13Rα1 peptide compared to thebinding between said antibody and the hIL-13Rα1 peptide without saidsubstitution.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Production and Purification of a Recombinant Protein Based onthe Human IL-13Rα1 Extracellular Region

Using the protocol described herein, an N-terminal FLAG®-tagged fusionprotein including most of the extracellular region of human IL-13Rα1(amino acids number 3 to 317 of SEQ ID NO:101) was purified from culturemedia conditioned by a stably transfected (pEFBOS-S-FLAG® vectorencoding IL-13Rα1.ECR) CHO cell clone. The purified hIL-13Rα1.ECRprotein (SEQ ID NO:103) was concentrated and subsequently desalted intophosphate-buffered saline (PBS), 0.02% v/v TWEEN™ 20, followed by filtersterilization. Typical recovery was 0.4 mg protein per liter ofconditioned media. Protein was stored at −80° C. until required.

Methods.

A pEFBOS-S-FLAG® expression vector incorporating a cDNA encoding theextracellular region (ECR) of human IL-13Rα1 with an IL-3 signalsequence and FLAG®-tag fusion was transfected into CHO cells for stableexpression using standard procedures. CHO-derived conditioned media wasthen concentrated 10-fold by ultrafiltration using a 10 kDa cut-offmembrane. Concentrated media was applied to an M2 (anti-FLAG®) affinitychromatography column and eluted with FLAG® peptide at a concentrationof 100 μg/ml. The eluate was concentrated by lyophilization and thenbuffer exchanged into Tris pH 8.0 using GF50 SEPHAROSE™ resin packedinto column (2.6 cm×40 cm). The buffer exchanged fraction was thensubjected to anion exchange chromatography using a MONO™ Q 5/5 column. Aproteolysed fragment of IL-13Rα1 was strongly retained and was separatedfrom the full-length IL-13Rα1 which eluted at a lower saltconcentration. Pooled fractions and filter sterilized final product wasassessed by SDS-PAGE and western blot analysis. The sample was thenquantitated using UV absorbance, where 1 absorbance unit wasapproximately 1 mg/ml.

EXAMPLE 2 Generation of Hybridoma Cell Lines Producing Human Anti-HumanIL-13Rα1 Monoclonal Antibodies

Immunization of Transgenic Mice.

Male and female transgenic mice from the HCo7, HCo12 and HCo7xHCo12strains (HUMAB™ mice, Medarex, USA) were immunized with hIL-13Rα1.ECR ofExample 1. For the first immunization, 20-50 of hIL-13Rα1.ECR wasemulsified in Complete Freund's Adjuvant (CFA) and administered via theintraperitoneal (i.p.) route. For a minimum of two and a maximum ofthree subsequent i.p. immunizations, 20-50 μg of hIL-13Rα1.ECR wasemulsified in Incomplete Freund's Adjuvant (IFA). Following the secondor third immunization with hIL-13Rα1.ECR in IFA, serum was sampled(retro-orbital plexus) and assayed for human antibodies against thehIL-13Rα1.ECR by ELISA as described herein. High-responder mice (serumtiters generally >1:3200) were selected for hybridoma generation. Insome cases, animals not used for hybridoma generation at this pointreceived further i.p. immunizations with 20-50 μg of hIL-13Rα1.ECR inPBS. Serum from these animals was again assayed for human antibodiesagainst the hIL-13Rα1.ECR by ELISA and high-responder mice were used forhybridoma generation. Mice selected for hybridoma generation wereboosted intravenously with 20-50 μg of hIL-13Rα1.ECR 3-4 days prior tospleen cell fusion.

Antigen-Specific ELISA.

Mouse serum or hybridoma culture supernatant fluid (SNF) was assessedfor mAbs able to bind to plate bound hIL-13Rα1.ECR using a standardELISA format as follows. Flat bottom 96-well MAXISORP™ plates (NUNC,Invitro Technologies, #439-454) were coated with 50 μl of a solutioncontaining 2.5 μg/ml hIL-13Rα1.ECR diluted in PBS and incubatedovernight at 4° C. After washing two times with PBS, plates were blockedwith 2% w/v skim milk in PBS (blocking buffer, 200 μl/well) for 1 hourat 37° C., then washed a further two times with PBS containing 0.1% v/vTWEEN™ 20 (wash buffer). Fifty μl of test hybridoma SNF or mouse serumwas added per well and plates were incubated at room temperature for 1hour. Plates were washed three times. Bound human mAbs were detectedusing an anti-human IgG HRP-conjugated secondary reagent diluted 1:1000in PBS containing 1% w/v skim milk powder and 0.1% v/v TWEEN™ 20. Fiftyμl/well of the anti-human IgG HRP-conjugated secondary reagent was addedto the plates for 1 hour at room temperature. The plates were thenwashed three times, developed with TMB substrate, and OD read at 450 nm.

Hybridoma Generation.

Selected high-responder mice were sacrificed and the spleen and relevantlymph nodes were collected. The fusion of spleen and lymph node cellswith the fusion partner SP2/O and subsequent HAT(hypoxanthine/aminopterin/thymidine) (GIBCO-BRL, #21060-017) selectionof hybridomas were performed according to standard procedures(Antibodies: A Laboratory Manual, Harlow and Lane, Cold Spring HarborLaboratory Press). Briefly, the centrifuge was adjusted to roomtemperature, with a water bath to 37° C. and a heat block to 37° C.Polyethylene glycol (PEG) was warmed to 37° C. Medium was prepared forculturing cells after the fusion was completed. The medium includedhybridoma serum-free medium (HSFM)(GIBCO-BRL, #12045-084), 5% Ultra lowIgG FBS (FBS) (GIBCO-BRL, #16250-078), 2 mM Glutamax-1 (GIBCO-BRL,#35050-061), 50 U/50 μg/ml Penicillin/Streptomycin (GIBCO-BRL,#15070-063) and 1×HAT. Media was warmed to 37° C. SP2/O cells wereharvested and a viable cell count was performed. The cells were healthy,actively dividing and in log-phase. The viability was >95%. SP2/Os werecultured in HSFM/5% Ultra low IgG FBS prior to fusion, and split 1:2 or1:3 on the day before the fusion. On the day of fusion, the animals weresacrificed and the spleen (and lymph nodes if required) were immediatelyremoved into sterile medium (Dulbecco's modification of Eagles media(GIBCO-BRL, #11995-073) or DME) on ice. A single-cell suspension wasprepared from the spleen, and washed twice (1800 rpm for 7 minutes) inDME, the second wash warm. The SP2/O cells were washed three times (1500rpm, 7 minutes) with warm DME to remove all traces of serum. SP2/O cells(10⁸) were used for one mouse spleen, done as two separate fusions.SP2/Os and spleen cells were pooled together in the same tube andcentrifuged at 2100 rpm (400 g) for 5 minutes. All DME was removed,leaving only combined pellet. The DME was placed in 37° C. heat block.One ml of warm PEG was added drop-wise to the cell pellet over 1 minutewhilst stirring the pellet gently with the pipette. Stirring continuedgently for another minute. One ml warm DME was added, drop-wise,stirring, over 1 minute. Another 1 ml DME was added over 1 minute. Then20 ml DME was added over 5 minutes while stirring slowly. This was thencentrifuged for 5 minutes at 1500 rpm. All supernatant was removed, andcells were resuspended gently in culture medium as above. One mousespleen was plated to 5 microtiter plates at 0.2 ml per well in HATmedium. The plates were fed by removing approximately 0.1 ml from eachwell and replacing with fresh HAT medium every 3 or 4 days. Wells werechecked for growth of hybridomas at day 7-10 (routine screening 10-14days after the fusion). Being sure that the medium had not been changedfor at least 2-3 days beforehand, ˜100 μl of supernatant was removedfrom each well for assay. Positives were transferred to 1 ml or 2 mlwells then gradually expanded to 6-well plates. Hybridomas were notclonal at this stage. After 14 days in HAT medium, hybridomas werecultured in HT (GIBCO-BLR, #11067-030) (HSFM, 5% Ultralow IgGFBS, 10ng/ml rhIL-6 (R&D Systems, #206-IL-050) and HT) for approximately 2 moreweeks, then without HT.

Culture of Hybridomas.

Hybridomas testing positive at primary and follow-up confirmation ELISAscreens were cloned by limit dilution. Limit dilution wells containingsingle colonies were screened by ELISA and a positive well was selectedfor expansion. Further rounds of limit dilution cloning were carried outuntil 100% of wells test positive.

For production of supernatant fluid (SNF) for antibody purification,hybridomas were expanded into either T175 cm² flasks (Falcon, #3028) orroller bottles (900 cm²) (CORNING, #430849). Media used for generationof hybridoma SNFs was HSFM supplemented with 5% Ultralow IgG FBS, 2 mMglutamine and 50 U/50 μg/ml penicillin/streptomycin. Hybridomas wereallowed to grow to confluence and media was harvested by centrifugationapproximately 5-10 days later when >90% of cells were dead. Allconditioned media was filtered using a STERICUP™ filter apparatus(MILLIPORE, #SCGPU11RE) (0.45 μm) prior to mAb purification.

Production of Purified mAbs.

Monoclonal antibodies were purified from SNF using a standard Protein Aaffinity chromatography based strategy; see e.g., the followingapparatus and reagents. HPLC: AKTA explorer (AMERSHAM Biosciences,Sweden); Column: Protein A (HITRAP™, 1 ml, Amersham Biosciences,Sweden); Buffer A: PBS, 0.02% TWEEN™ 20; Buffer B: 0.1 M Glycine pH 2.8;and Buffer C: 2 M Tris pH 8.0.

The column was prepared by washing with 5 volumes of buffer A.Conditioned media was loaded onto dedicated column. A wash was performedwith 100 volumes of buffer A, and elution with 20 ml (10×2 ml) of bufferB. Collection was into tube containing 0.2 ml of buffer C. Column waswashed with buffer A to store at 4° C. Desalting was performed using 10Kcut-off dialysis membrane into PBS, 0.02% TWEEN™ 20. mAb purity wasdemonstrated by SDS-PAGE with COOMASSIE® Blue staining.

Antibody was quantitated by spectrophotometric analysis at 280 nm usingan immunoglobulin extinction coefficient of 1.0 absorbance unit beingequivalent to 1.34 mg/ml of antibody.

EXAMPLE 3 Analysis of Anti-Human IL-13Rα1 Monoclonal Antibody 10G5Affinity for Human IL-13Rα1

BIACOR™-Based Studies.

Human IL-13Rα1.ECR (40 μg/ml in 20 mM Sodium Acetate, pH 4.2) of Example1 was immobilized to a sensorchip (CM5, Biosensor, Sweden) usingstandard NHS/EDC chemistry according to the manufacturer's instructionsat a set immobilization value, for example, 1000 RU. Ethanolamine (1.0M), pH 8.0 was used to quench residual active esters post hIL-13Rα1.ECRimmobilization.

Analysis of binding of 10G5 (concentration range of 1.4 nM to 150 nM,two-fold dilutions) to the immobilized hIL-13Rα1.ECR was performed induplicate. Sensorgrams generated were fitted to a bivalent ligandbinding model to simultaneously derive association (k_(a)) anddissociation (k_(d)) rates and used to determine binding affinity(K_(D), Biaevaluation software, BIACORE™, Sweden).

The binding affinity (K_(D)) of anti-IL-13Rα1 human mAb 10G5 was ˜254 pM(n=8).

EXAMPLE 4 Analysis of the Binding of Anti-Human IL-13Rα1 MonoclonalAntibody 10G5 to Cynomolgus Macaque and Mouse IL-13Rα1

A cDNA encoding the cynomolgus macaque IL-13Rα1 (cyIL-13Rα1) was clonedby PCR using mRNA extracted from cynomolgus spleen and bone marrow. Themature sequence was highly conserved between cynomolgus and humanIL-13Rα1 with an amino acid identity of about 97% (see GENBANK accessionNo. AAP78901).

For production of purified cynomolgus IL-13Rα1.ECR protein, a cDNAencoding cynomolgus IL-13Rα1.ECR (amino acids 9 to 325 of GENBANKaccession No. AAP78901 or amino acids 1 to 317 of SEQ ID NO:104) wascloned into the pEFBOS-S-FLAG® vector for expression as an N-terminalFLAG®-tagged fusion protein essentially as described above for thehIL-13Rα1.ECR.

Mouse IL-13Rα1.ECR (amino acids 27 to 344 of GENBANK accession No.009030 or amino acids 1 to 318 of SEQ ID NO:105) was also expressed andpurified as an N-terminal FLAG®-tagged fusion (mIL-13Rα1.ECR)essentially as described above.

The potential cross-reactivity of mAb 10G5 with mouse and cynomolgusIL-13Rα1.ECR was assessed using a BIACORE™-based approach. Purifiedmouse, human and cynomolgus IL-13Rα1.ECR were immobilized individuallyto three channels of a sensorchip (CM5, BIACORE™, Sweden) using standardimmobilization chemistry. Monoclonal antibodies (concentration range of312.5 nM down to 125 pM) were assessed for binding to the receptorssimultaneously at a flow rate of 15 μl/minute. Analysis of the affinityof the mAb was performed as described in Example 3 above.

The results of this analysis indicated that MAb 1065 showed a 10-foldlower affinity (˜2.9 nM) for the cynomolgus receptor compared with thehuman receptor (˜254 pM), and negligible binding to mouse receptor.

EXAMPLE 5 Analysis of the Ability of 10G5 to Compete with IL-13 forBinding of Human IL-13Rα1

The ability of 10G5 to compete with IL-13 for binding to IL-13Rα1 wasassessed by a competition assay on a BIACORE™ instrument. A sensorchipwas prepared by immobilizing human IL-13 using standard NHS/EDCchemistry as per the manufacturer's instructions. hIL-13Rα1.ECR protein(8 μg/mL) was incubated with excess mAb (50 μg/mL) for 2 hours at roomtemperature, then injected over the sensorchip. The level ofhIL-13Rα1.ECR protein bound to immobilized IL-13 was recorded at a fixedtime point within the sensorgram and divided by the corresponding levelof hIL-13Rα1.ECR protein bound in the absence of mAb (“relative IL-13binding”). Binding ratios <1 were indicative of competition between mAband IL-13 for binding to IL-13Rα1, while values 1 indicated the mAbbound to IL-13Rα1 at a site distinct from that binding IL-13.

mAb 10G5 was found to inhibit binding of the hIL-13Rα1.ECR protein toimmobilized IL-13.

EXAMPLE 6 Analysis of the Ability of Anti-Human IL-13Rα1 MonoclonalAntibodies to Antagonize IL-13- and IL-4-Mediated Cellular Responses

Normal Human Dermal Fibroblast (NHDF) Eotaxin Assay.

NHDF cells have been demonstrated to produce eotaxin in response toIL-13 and mAbs directed against the IL-13Rα1 may inhibit this response.Furthermore, NHDF cells were demonstrated not to express the γcreceptor, thus enabling an analysis of the ability of mAbs to inhibitIL-4 activity mediated through the IL-4 Type II receptor, i.e.,IL-4Rαplus IL-13Rα1. Due to species cross-reactivity, both human andnon-human primate (e.g., rhesus) IL-13 can be used to stimulate eotaxinproduction.

NHDF cells (Cambrex, #CC-2509) were cultured in FGM media (Cambrex,#CC3132) supplemented with the recommended additives according to themanufacturer's instructions (complete media). Cells were passaged 1:3 or1:5 once a week and monitored for responsiveness to IL-13 prior to use.To assess antagonist activity of hIL-13Rα1 specific mAbs, cells wereresuspended to 2×10⁶/ml in complete media containing 20 ng/ml PMA(SIGMA, #P8139) and 20 μg/ml polymyxin (SIGMA, #P4932) and plated in96-well flat bottom plates (COSTAR, #3595) at 1×10⁵ cells/well. Antibodytitrations were added to the cells and incubated for 30 minutes, at 37°C. with 5% CO₂ in humidified air. Recombinant IL-13 (human or non-humanprimate) was then added to plates at a final concentration of 30 ng/mland incubated overnight at 37° C. with 5% CO₂ in humidified air.Supernatants were then removed and assayed for eotaxin content by ELISA.For IL-4-induced assays, recombinant IL-4 (PHARMINGEN) was added toplates at a final concentration of 0.5 ng/ml in place of IL-13.

Eotaxin ELISA Protocol.

IMMULON®-4 plates (DYNATECH, #3855) were coated with 4 μg/ml mouseanti-human eotaxin antibody (R&D Systems, MAB320) in PBS (INVITROGEN,#14190-144), overnight at 4° C. Plates were blocked (200 μl/well, TBSsupplemented with 1% BSA and 0.05% TWEEN™ 20) for 1 hour at roomtemperature and washed three times (wash buffer, TBS plus 0.05% TWEEN™20). Test SNF's from the NHDF cells were added at 50 μl/well and plateswere incubated for 2 hours at room temperature, then washed three times.Biotinylated anti-human eotaxin antibody (R&D Systems, BAF320) was addedat 200 ng/ml in blocking buffer (60 ml/well) and incubated for 1 hour atroom temperature, then washed three times. Streptavidin-Europium(Wallac, #1244-360) was added at 100 ng/ml in europium buffer (100μl/well) and incubated for 20 minutes at room temperature, then washedthree times. Enhancement solution (Wallac, #12244-105) was added, 150μl/well, and incubated 1 hour at room temp. Time delayed fluorescencewas read using a VICTOR (PERKIN-ELMER) plate reader.

Recombinant human eotaxin (R&D Systems, #320-EO) was used to establish astandard curve. The results of this analysis indicated that the EC₅₀ of10G5 against IL-13 was 0.25 μg/ml, whereas the EC₅₀ against IL-4 was 2.7μg/ml.

NHDF IL-13/IL-4-Induced STAT6 Phosphorylation Assay.

The phosphorylation of STAT6 (pSTAT6) is an essential element ofIL-13/IL-4 signal transduction and occurs within minutes of receptordimerization. IL-13Rα1-specific mAbs may block the phosphorylation ofSTAT6 in response to IL-13 and/or IL-4.

To determine this, 2×10⁶ NHDF cells in 50 μl of RPMI media (#22400-071,INVITROGEN) were plated into 96-well V bottom polypropylene PCR plates(USA Scientific, #1442-9596). Anti-IL-13R mAbs were added to therequired concentration in 25 μl and plates were incubated for 30 minutesat 4° C. Recombinant hIL-13 (100 ng/ml) or hIL-4 (PHARMINGEN, 0.5 ng/ml)was added in 25 μl and plates were warmed to 37° C. in a PCR machine for20 minutes. After 20 minutes an equal volume of 2× lysis buffer (100 mMHEPES, 200 mM NaCl, 2% v/v TRITON® X100, 100 mM NaF, 10 mM DTT, proteaseinhibitors) was added and pSTAT6 was measured by ELISA.

STAT6 ELISA Protocol.

IMMULON®-4 plates (DYNATECH, #3855,) were coated with anti-human phosphoSTAT6 (BD Transduction Labs, #621995) at 10 μg/ml in PBS (INVITROGEN,#14290-144) (50 μl/well) and incubated overnight at 4° C. Plates wereblocked (200 μl/well, TBS supplemented with 1% BSA and 0.05% TWEEN™ 20)for 1 hour at room temperature and washed three times (wash buffer, TBSplus 0.05% v/v TWEEN™20). Test lysates were added at 50 μl/well andplates were incubated for 2 hours at room temperature, then washed threetimes. Biotin anti-STAT6 (BD Transduction Labs, #621141, conjugated tobiotin, 20:1 molar ratio) was added at 2 μg/ml in blocking buffer (60μl/well) and incubated for 1 hour at room temperature, then washed threetimes. Streptavidin-Europium (Wallac, #1244-360) was added at 100 ng/mlin europium buffer (100 μl/well) and incubated for 20 minutes at roomtemperature, then washed three times. Enhancement solution (Wallac,#12244-105) was added (150 μl/well) and incubated 1 hour at roomtemperature. Time-delayed fluorescence was read using a VICTOR(PERKIN-ELMER) plate reader.

The results of this analysis indicated that the EC₅₀ of 10G5 was 1.0μg/ml against IL-13, whereas the EC₅₀ against IL-4 was 1.3 μg/ml.

EXAMPLE 7 Cloning and Sequencing of the 10G5 Murine Antibody VariableRegions

Messenger RNA was prepared from hybridoma cells producing antibody 10G5and reverse-transcribed using an oligo-dT primer to produce cDNA.Several independent PCR reactions were performed. The PCR reactions wereperformed at the following conditions: 94° C. for 2 minutes; 30 cyclesof 94° C. for 30 seconds, 60° C. for 30 seconds and 68° C. for 1 minute;and 68° C. for 10 minutes. Two alternative PCR conditions were alsodeveloped in cloning antibody genes: 1) 94° C. for 2 minutes; 30 cyclesof 94° C. for 30 seconds, 68° C. for 30 seconds, and 68° C. for 1minute; and 68° C. for 10 minute; and 2) 30 cycles of 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 1 minute. PCR amplicons wereseparated on 1.2% agarose gels. In terms of the heavy chain variableregion, the following primer sets yielded a PCR product. For the 5′ endof the heavy chain variable region, the primers were VH5,5′-G GGG TCAACC GCC ATC CTY G-3′ (SEQ ID NO:114), wherein Y was C or T (Degen 2);and VH6,5′-GTC TCC TTC CTC ATC TTC CTG CCC-3′ (SEQ ID NO:115) (Degen 1);while the primer for the 3′ end of V_(H) was HA, 5′-C CCA TCG GTC TTCCCC CTG GCA C-3′ (SEQ ID NO:116). In terms of the light chain variableregion, four primer sets yielded a PCR product. The PCR products werecloned in plasmid TOPO® pCK2.1 (INVITROGEN). Sequence analysis of theheavy chain clone had the best match to germline, VH5-51. For the lightchain variable region, the primers for the 5′ end that yielded asequence with the best match to germline, VL VKIII A27, were VK3,5′-YTCTTC CTC CTG CTA CTC TGG CTC-3′ (SEQ ID NO:117), wherein Y was C/T (Degen2); and VK4,5′-ATG GTG TTG CAG ACC CAG GTC TTC-3′ (SEQ ID NO:118) (Degen1); while the primer for the 3′ end of V_(L) was KA, 5′-G AAA TCT GGAACT GCC TCT GTT GTG TGC CTG C-3′ (SEQ ID NO:119).

The initially identified nucleotide and amino acid sequences containingthe heavy and light chain variable regions of 10G5 included both leaderand some additional downstream sequence (see, SEQ ID NOs:42 and 44 forthe heavy chain, and SEQ ID NOs:46 and 48 for the light chain). Thenucleotide and amino acid sequences of the heavy chain variable regionof 10G5 are shown in FIG. 1 and in SEQ ID NOs:43 and 45, respectively.The nucleotide and amino acid sequences of the light chain variableregion of 10G5 are shown in FIG. 2 and in SEQ ID NOs:47 and 49,respectively.

EXAMPLE 8 Analysis of mAb Binding to Phage Displayed Human IL-13Rα1Peptides

Mutation of the phenylalanine at position 233 of the amino acid sequenceof human IL-13Rα1 extracellular region to alanine effected a significantdecrease in binding of antibody 10G5 as compared to binding of theantibody to wild-type human IL-13Rα1 extracellular region. Accordingly,Phe233 was identified as an important residue on the receptor forbinding of antibody 10G5. Conveniently, the human IL-13Rα1 extracellularregion or the corresponding Phe233Ala mutant were fused via theC-terminus to a fragment of the gene 3 protein (amino acids 249-406)generally in accordance with the procedure described by Lowman et al.,1991 Biochem. 30:10832-10838. These IL-13Rα1-derived peptides were thendisplayed on M13 bacteriophage and assayed by ELISA for binding toimmobilized 10G5 or 1D9. Briefly, mAbs were passively adsorbed onto96-well MAXISORP™ plates (NUNC) following overnight incubation of 100μl/well of 2.5 μg/mL mAb diluted in PBS buffer. Coating solutions werediscarded, plates were blocked by incubation with blocking buffer for 1hour at room temperature, then washed once with wash buffer. Phagesamples serially diluted with 1% w/v skim milk powder in PBS (dilutingbuffer) were then transferred into mAb-coated plates (100 μL/well).Following incubation at room temperature for 2 hours, plates were washed3 times, and bound phage labeled with anti-M13 IgG HRP-conjugatedpolyclonal antibody, and detected by addition of TMB substrate. TMBcolor development was quenched by addition of 2 M aqueous sulfuric acid,and absorbance at 450 nm was measured.

EXAMPLE 9 Antibody Identification

Methods.

The variable heavy and the variable light sequences of antibody 10G5(SEQ ID NOs:43 and 47, respectively) were cloned in a Fab phage-displayvector, pFab3d (FIGS. 3, 4A, and 4B) with a 1929 bp XhoI/ApaI fragmentfrom the PKS3 locus of the fungus Glarea lozoyensis cloned at theXhoI/ApaI site as a stuffer in the light chain construct, then randomlymutated in the variable heavy and light CDR3 regions (each librarypossessing ≧10⁸ functional diversity). The resultant mutants were thenpanned against biotinylated human and primate (rhesus and cynomologousmonkey) IL-13Rα1 in solution using standard phage display protocols(see, e.g., Phage Display: A Laboratory Manual, 2001, Cold Spring HarborLaboratory Press). Human and primate sequences have been disclosed inthe literature; see, e.g., GENBANK Accession Nos. U62858, CAA70508, andAAP78901. By lowering the concentration of target in each subsequentround of panning (e.g., 10 nM, 1 nM, 0.1 nM, and 0.01 nM), thestringency of panning was effectively increased, thereby enriching forhigher and higher affinity phage with each subsequent round. Phage ELISAwas used as the primary assay to determine the ability of thephage-bound recombinant Fabs to recognize the biotinylated IL-13Rα1immobilized on streptavidin plates (see, e.g., Phage Display: ALaboratory Manual, supra). Myc-capture ELISA and dissociation assays(general protocols described herein) were used as secondary screeningtools. BIACORE™ surface plasmon resonance and KINEXA™ kinetic exclusionassays were performed to characterize the binding kinetics of theantibodies identified. These assays were performed in accordance withthe published manufacturers' protocols. Specific antibodies wereconverted into full-length antibodies of subclass IgG4 for expression,production and characterization in mammalian cells (general protocoldescribed below).

Myc Capture and Dissociation Assays.

Two assays are run in parallel. The first (I) measured the amount ofantibody captured from peripreps. This assured that data was collectedonly from wells that had sufficient and equivalent amounts of antibody.The second (II) measured the dissociation of IL-13 receptor from theplate-bound antibody.

Assay (I):

IMMULON®-4 plates (DYNATECH, #3855) were coated with polyclonalanti-human kappa antibody (Immunology Consultants Lab, #GKBF-80A-K116)at 5 μg/ml in PBS (INVITROGEN, #14290-144) (50 μl/well) and incubatedovernight at 4° C. Blocking buffer (200 μl/well) was added and theplates were incubated for 1 hour at room temperature. The plates werewashed three times with wash buffer. Neat periprep was added, 50μl/well, and left for 2 hours at room temperature. The plates werewashed three times with wash buffer. Fifty μg/ml of human gamma globulin(Pierce, #31879) was added in block buffer and left to incubateovernight at 4° C. The plates were washed three times with wash bufferin the morning and afternoon followed by the addition of 150 μl/well ofblock buffer while incubating at 37° C. throughout. The plates werewashed three times with wash buffer. Bound antibody was detected withbiotin anti-Myc (Upstate, #16-212) at 1 μg/ml in blocking buffer (60μl/well) for 1 hour at room temperature. The plates were washed threetimes with wash buffer. Streptavidin-europium (Wallac, #1244-360) wasadded at 100 ng/ml in Europium buffer (100 μl/well) for 20 minutes atroom temperature. A final wash step (three times) was performed andenhancement solution (Wallac, #1244-105) at 150 μl/well was added for 1hour at room temperature. Plates were read by time-delayed fluorescenceon a VICTOR (PERKIN-ELMER) plate reader.

Assay (II):

IMMULON®-4 plates (DYNATECH, #3855) were coated with polyclonalanti-human kappa antibody (Immunology Consultants Lab, #GKBF-80A-K116)at 5 μg/ml in PBS (INVITROGEN, #14290-144) (50 μl/well) and incubatedovernight at 4° C. Blocking buffer (200 μl/well) was added and theplates were incubated for 1 hour at room temperature. The plates werewashed three times with wash buffer. Neat periprep was added, 50μl/well, and left for 2 hours at room temperature. The plates werewashed three times with wash buffer. Sixty μl/ml of 400 ng/mlFLAG®-tagged human IL13 receptor was added with 50 μg/ml of human gammaglobulin (Pierce, #31879) in block buffer and left to incubate overnightat 4° C. The plates were washed three times with wash buffer at two,six-hour intervals followed by the addition of 150 μl/well of blockbuffer. Incubations were carried out at 37° C. The plates were washedthree times with wash buffer. Residual IL-13 receptor was detected withbiotin anti-FLAG® (IBI, #3081/6H2411) at 1 μg/ml in blocking buffer (60μl/well) for 1 hour at room temperature. The plates were washed threetimes with wash buffer. Streptavidin-europium (Wallac, #1244-360) wasadded at 100 ng/ml in Europium buffer (100 μl/well) for 20 minutes atroom temperature. A final wash step (three times) was performed andenhancement solution (Wallac, #1244-105), 150 μl/well, was added for 1hour at room temperature. Plates were read by time-delayed fluorescenceon a Victor (Perkin-Elmer) plate reader.

Conversion to Full-Length IgGs.

Anti-IL13Rα1 monoclonal antibodies were converted into whole antibody ofsubclass IgG4 for expression and production in mammalian cells. Theirvariable regions were PCR-amplified from the corresponding Fab vectorsand in-frame cloned into a LONZA pCON antibody expression vector withleader sequences in front of the antibody sequences. In the vector,genomic DNA sequences for all constant regions for light and heavychains were already engineered in the vectors. The expression is drivenby a human cytomegalovirus (CMV) early promoter and followed by an SV40polyadenylation signal. The plasmids have bacterial sequence for plasmidreplication and ampicillin selection marker and the plasmid for thelight chain, pCONKAPPA, has the GS gene for glutamine synthetase as aselection marker in mammalian cells. In-frame fusion of variable regionsallows the proper expression of whole antibody. By design, leadersequences from mouse light and heavy chains were included in front ofthe antibody open reading frames. A consensus Kozak sequence (italicsonly) was also included surrounding the ATG start codon to improveprotein expression level. Forward and reverse primers were designed forPCR amplification of four variable regions. For 10G5 light chainvariable region, forward primer, 5′-ATC GAA GCT TGC CGC CAC CAT GAG TGTGCC CAC TCA GGT CCT GGG GTT GCT GCT GCT GTG GCT TAC AGA TGC CAG ATG TGAAAT TGT GTT GAC GCA GTC T-3′ (SEQ ID NO:88) and reverse primer, 5′-CCACCG TAC GTT TGA TTT CCA C-3′ (SEQ ID NO:89) were employed. For 10G5heavy chain variable region, forward primer, 5′-ACT GAA GCT TGC CGC CACCAT GGA ATG GAG CTG GGT CTT TCT CTT CTT CCT GTC AGT AAC TAC AGG TGT CCACTC CGA GGT GCA GCT GGT GCA GTC T-3′ (SEQ ID NO:90) and reverse primer,5′-ACC GAT GGG CCC TTG GTG GAG GCT-3′ (SEQ ID NO:91) were employed. Theleader sequences are in bold and underlined and the cloning sites(HindIII in the forward primers for both light and heavy chains, in thereverse primers, BsiWI for the light chain and ApaI for the heavy chain)are given in underlining and italics.

The variable regions were PCR-amplified for 20 cycles using these pairsof primers and Fab vectors carrying 10G5 variable region sequences. PCRproducts were digested with HindIII and BsiWI for light chains andHindIII and ApaI for heavy chains. Enzyme-digested PCR fragments werecloned into Lonza's vectors (pCONKAPPA for light chain and pCONGAMMA4for heavy chain). The entire expression cassette of respective heavychain from pCONGAMMA4 vectors digested with NotI and SalI was theninserted into the corresponding light chain vector digested with thesame enzymes. The entire open reading frames for both light chain andheavy chain were verified by DNA sequence analysis.

Antibody Expression, Purification and Characterization.

Either combined light chain and heavy chain plasmid DNA or a 1:1 ratiomixture of corresponding light and heavy chain plasmid DNA weretransfected in 293-derived cell lines. For pCON vectors, 293 FREESTYLE™suspension cell line from INVITROGEN was used along with itstransfection reagents. For 200 ml of 293 FREESTYLE™ cells, 100 μg eachof heavy and light chain plasmid DNA and 300 μl of reagents were usedfor transfection. The transfected cells were incubated at 37° C./5% CO₂for 7-8 days before harvest. Culture medium was harvested, filtered andconcentrated using by low speed MILLIPORE CENTRICON® centrifugation(concentrator, MILLIPORE).

Results.

ELISA analyses are presented in FIGS. 5-8. In FIGS. 5-7, the mutantssignificantly distinguished by their activity were 10G5H6 (VHCDR3: SEQID NO:5; VLCDR3: SEQ ID NO:41); 10G5H5 (VHCDR3: SEQ ID NO:4; VLCDR3: SEQID NO:41); 10G5H11 (VHCDR3: SEQ ID NO:3; VLCDR3: SEQ ID NO:41); and10G5H33 (VHCDR3: SEQ ID NO:25 (involving a The=>Ile residue change inthe third to last amino acid residue therein); VLCDR3: SEQ ID NO:41).FIG. 8 illustrates ten mutants highlighted as functioning moreeffectively than 10G5H6. The mutants tested in FIG. 8 have EC₅₀ valuesand possess the heavy and light CDR3 regions as listed in Table 4.

TABLE 4 Antibody VHCDR3 VLCDR3 EC₅₀ (μM) SJ2-66 SEQ ID NO: 5 SEQ ID NO:28 1.757 10G5H6 SEQ ID NO: 5 SEQ ID NO: 41 1.636 SJ2-88 SEQ ID NO: 5 SEQID NO: 27 1.536 SJ3-50 SEQ ID NO: 5 SEQ ID NO: 29 1.341 SJ2-74 SEQ IDNO: 5 SEQ ID NO: 32 1.308 SJ2-63 SEQ ID NO: 5 SEQ ID NO: 33 1.248 SJ3-68SEQ ID NO: 5 SEQ ID NO: 34 1.24 SJ3-71 SEQ ID NO: 5 SEQ ID NO: 35 1.186SJ2-57 SEQ ID NO: 5 SEQ ID NO: 36 1.14 SJ2-85 SEQ ID NO: 5 SEQ ID NO: 371.123 SJ2-81 SEQ ID NO: 5 SEQ ID NO: 38 0.9334

KINEXA® analyses were performed on select antibodies. The data forspecific full-length antibodies is illustrated in the Tables 5 and 6containing data from two different experiments.

TABLE 5 Antibody VHCDR3 VLCDR3 K_(D) 10G5-1 SEQ ID NO: 5 SEQ ID NO: 3854.84 pM 10G5-2 SEQ ID NO: 22 SEQ ID NO: 38 45.44 pM 10G5-4 SEQ ID NO:23 SEQ ID NO: 38 66.93 pM

TABLE 6 Antibody VHCDR3 VLCDR3 K_(D) 10G5 WT SEQ ID NO: 40 SEQ ID NO: 41  861 pM 10G5H6 SEQ ID NO: 5 SEQ ID NO: 41 99.43 pM 10G5-2 SEQ ID NO: 22SEQ ID NO: 38 31.44 pM 10G5-4 SEQ ID NO: 23 SEQ ID NO: 38 20.35 pM10G5-6 SEQ ID NO: 7 SEQ ID NO: 38  26.8 pM

BIACORE™ analyses were performed on various antibodies in Fab format.Tables 7 and 8 illustrate data from two different experiments:

TABLE 7 Antibody VHCDR3 VLCDR3 K_(D) 10G5 WT SEQ ID NO: 40 SEQ ID NO: 414.5 nM 10G5H6 SEQ ID NO: 5 SEQ ID NO: 41 0.6 nM

TABLE 8 Antibody VHCDR3 VLCDR3 K_(D) 10G5 WT SEQ ID NO: 40 SEQ ID NO: 411.35 nM   10G5H11 SEQ ID NO: 3 SEQ ID NO: 41 106 pM 10G5R4-10 SEQ ID NO:15 SEQ ID NO: 41 196 pM 10G5R4-11 SEQ ID NO: 16 SEQ ID NO: 41 233 pM10G5R4-2A SEQ ID NO: 7 SEQ ID NO: 41 817 pM 10G5R4-2B SEQ ID NO: 23 SEQID NO: 41 116 pM 10G5SJ2-81 SEQ ID NO: 5 SEQ ID NO: 38  43 pM

The data provided illustrate that antibodies could be identified,through the various screens and analyses conducted, with significantlyenhanced affinity for IL-13Rα1. The antibodies uncovered demonstratedquite frequently a general consensus in their sequence. Morespecifically, it was concluded from these studies that (1) threeresidues in the heavy chain variable region CDR3 domain were more apt topositively effect function upon mutation, namely, residues 1, 7 and 9 ofSEQ ID NO:40, and (2) three residues in the light chain variable regionCDR3 domain were more apt to positively effect function upon mutation,namely, residues 2, 4 and 5 in SEQ ID NO:41.

Antibodies with a manipulated Fc region were also developed. Themanipulations in the Fc region allowed for an antibody that exhibitsreduced binding to FcγR receptors or C1q. Binding to FcRn (also known asthe neonatal receptor or Brambell receptor) is not substantiallymodified nor is the antibody half-life. The purpose of the modificationswas to generate antibodies that do not provoke (or provoke to a lesserextent) antibody-dependent cellular cytotoxicity (ADCC),complement-mediated cytotoxicity (CMC), or form immune complexes, whileretaining normal pharmacokinetic (PK) properties. As will beacknowledged by the skilled artisan, the disclosed human antibody IgGstructure (that encompasses SEQ ID NO:92) can be used in conjunctionwith any variety of V_(H) and V_(L) sequences disclosed herein, with theappropriate Cκ or Cλ, in the development of full-length antibodies.

KINEXA® analyses were performed on select antibodies with themanipulated Fc. Amino acid and nucleotide sequences for 10G5-6 in themanipulated IgG format are set forth in SEQ ID NOs:94 and 95,respectively. Table 9 summarizes data obtained for select full-lengthantibodies upon 2-curve K_(d) analysis using KINEXA®.

TABLE 9 Antibody VHCDR3 VLCDR3 K_(D) 10G5-6 IgG4 SEQ ID NO: 7 SEQ ID NO:38 44.36 pM 10G5-6 IgG2m4 SEQ ID NO: 7 SEQ ID NO: 38 30.79 pM 10G5 WTIgG2m4 SEQ ID NO: 40 SEQ ID NO: 41  2.06 nM 10G5H6 IgG2m4 SEQ ID NO: 5SEQ ID NO: 41 94.64 pM

Antibodies manipulated in this manner have been found to exhibit severaladvantages over the native IgG isotypes. The first is that they do notbind C1q as strongly as IgG2, rendering it less effective in activatingthe complement cascade. The manipulated antibodies also do not bind, orexhibit significantly reduced binding, to Fcγ receptors atphysiologically relevant levels, in particular FcγRI, which eliminates(or significantly reduces) any undesired NK-cell or T-cell activation;significantly impedes the antibody's ability to mediate ADCC; andeliminates (or significantly reduces) a potential alternative sink forthe antibody in vivo. The resultant antibodies also retain the half-lifeand basic structure of an IgG2, which is highly desirable. The bloodhalf-life of 10G5-6 IgG2m4 was found in a separate study to be in theorder of 306 hours when tested in SCID mice. This half-life iscomparable with the numbers reported for IgG2; see, e.g., Zuckier etal., 1994 Cancer Suppl. 73:794-799.

EXAMPLE 10 Functional Studies—Eotaxin Release

NHDF Eotaxin Release Assay.

NHDF cells were purchased from Cambrex (#CC-2509) and were cultured inFGM media (Cambrex, #CC-3132) supplemented with additives provided,referred to below as complete media. Cells were passaged 1:3 or 1:5 oncea week and were monitored for responsiveness to IL-13 prior to use. Toassess antagonistic activity of IL-13Rα1 antibodies, cells wereresuspended to 2×10⁶/ml in complete media containing 20 ng/ml PMA(SIGMA, #P8139) and 20 μg/ml polymyxin (SIGMA, #P4932) and plated in96-well flat bottom plates (COSTAR, #3595) at 1×10⁵ cell/well. Antibodytitrations were added to the cells and incubated for 30 minutes at 37°C. in a 5% CO₂ incubator. Recombinant rhesus IL-13 or recombinant humanIL4 (BD PHARMINGEN, #554605) was then used at the respective EC₅₀ foreach cytokine and incubated overnight at 37° C. in a 5% CO₂ incubator.Supernatants were removed and assayed for eotaxin content byimmunoassay. Briefly, IMMULON®-4 plates (DYNATECH, #3855) were coatedwith 2 μg/ml anti-human eotaxin antibody (PHARMINGEN, #555035) in PBS(INVITROGEN, #14190-144) overnight at 4° C. The plates were blocked withblocking buffer for 1 hour at room temperature and washed three timeswith wash buffer. Supernatants from the NHDF cells were added to theplates along with a recombinant human eotaxin standard (R&D Systems,#320-EO). The samples were captured for 2 hours at room temperature,washed and biotinylated anti-human Eotaxin detection antibody(PHARMINGEN, #555060) was added at 200 ng/ml for 1 hour at roomtemperature. Plates were washed and streptavidin-europium (Wallac,#1244-360) was added at a concentration of 100 ng/ml for 20 minutes atroom temperature. A final wash step was performed and enhancementsolution (Wallac, #1244-105) was added for 1 hour at room temperature.Plates were read by time-delayed fluorescence on a VICTOR (PERKIN-ELMER)plate reader.

Results.

Eotaxin release from normal human dermal fibroblast (NHDF) cells uponcontact with IL-13Rα1-specific antibodies was analyzed. The assays werecarried out using either IL-13 and/or IL-4 as a stimulant. When IL-13was used as the inducing agent, the optimized antibodies were at least2-fold more potent in these types of assays than the parental form,10G5. An example of the fold-difference in functioning is illustrated inFIG. 9, where IC₅₀ values for 10G5, 10G5H6, and 10G5-6 were determinedto be 310 ng/mL (˜2 nM), 110 ng/mL (˜730 pM), and 70 ng/mL (˜467 pM),respectively, with IL-13 as the stimulant. FIG. 10 illustrates antibodyinhibition of the formation of eotaxin by NHDF cells where IL-4 is usedas the stimulant. An experiment was performed with a number offull-length antibodies of the present description. Data concerninginhibition of eotaxin release from NHDF cells upon stimulation by IL-13is summarized in Table 10.

TABLE 10 Antibody VHCDR3 VLCDR3 EC₅₀ 10G5 WT SEQ ID NO: 40 SEQ ID NO: 412.309 μg/ml 10G5-1 SEQ ID NO: 5 SEQ ID NO: 38 0.497 μg/ml 10G5-2 SEQ IDNO: 22 SEQ ID NO: 38 0.456 μg/ml 10G5-3 SEQ ID NO: 5 SEQ ID NO: 39 0.614μg/ml 10G5-4 SEQ ID NO: 23 SEQ ID NO: 38 0.330 μg/ml 10G5-5 SEQ ID NO:23 SEQ ID NO: 41 0.939 μg/ml 10G5H6 SEQ ID NO: 5 SEQ ID NO: 41 0.474μg/ml

Data concerning inhibition of eotaxin release from NHDF cells uponstimulation by IL-4 is summarized in Table 11.

TABLE 11 Antibody VHCDR3 VLCDR3 EC₅₀ 10G5 WT SEQ ID NO: 40 SEQ ID NO: 414.533 μg/ml 10G5-1 SEQ ID NO: 5 SEQ ID NO: 38 0.907 μg/ml 10G5-2 SEQ IDNO: 22 SEQ ID NO: 38 0.730 μg/ml 10G5-3 SEQ ID NO: 5 SEQ ID NO: 39 0.983μg/ml 10G5-4 SEQ ID NO: 23 SEQ ID NO: 38 0.660 μg/ml 10G5-5 SEQ ID NO:23 SEQ ID NO: 41 2.267 μg/ml 10G5H6 SEQ ID NO: 5 SEQ ID NO: 41 1.438μg/ml

EXAMPLE 11 Functional Studies—STAT6 Phosphorylation

NHDF STAT6 Phosphorylation Assay.

NHDF cells were purchased from Cambrex (#CC-2509) and were cultured inFGM media (Cambrex, #CC-3132) supplemented with additives provided.Plate NHDF cells at 2e6/ml in 50 μl volume in 96-well V-bottompolypropylene PCR plates (USA Scientific, #1442-9596) in RPMI Media(INVITROGEN, #22400-071). Anti-IL-13R antibodies were added in 25 μlvolume and incubated for 30 minutes at 4° C. Recombinant rhesus IL-13 orrecombinant human IL-4 (PHARMINGEN) was added in 25 μl volume. Theplates were warmed to 37° C. in PCR machine for 20 minutes and,immediately, equal volume of 2× lysis buffer (100 μl) was added. pSTAT6was measured by immunoassay. IMMULON®-4 plates (DYNATECH, #3855) werecoated with anti-human phospho STAT6 (BD Transduction Labs, 621995) at10 μg/ml in PBS (INVITROGEN, #14290-144,) (50 μl/well) overnight at 4°C. Blocking buffer (200 μl/well) was added for 1 hour at roomtemperature. The plates were washed three times with wash buffer. Fiftyμl/well lysate was added and incubated for 2 hours at room temperature.The plates were washed three times with wash buffer. Detection wasenabled with biotin anti-STAT6 (BD Transduction Labs, conjugated 20:1molar ratio) at 2 μg/ml in blocking buffer (60 μl/well) added for 1 hourat room temperature. The plates were washed three times with washbuffer. Streptavidin-Europium (Wallac, #1244-360) at 100 ng/ml was addedin europium buffer (100 μl/well) for 20 minutes at room temperature. Theplates were washed three times with wash buffer. Enhancement solution(Wallac, #12244-105) was added (150 μl/well) for 1 hour at roomtemperature, and plates were read by time-delayed fluorescence on aVICTOR (PERKIN-ELMER) reader.

Results.

Antibody inhibition of IL-13- and IL-4-induced STAT6 phosphorylation wasstudied in NHDF cells. When IL-13 was used as the inducing agent, theoptimized antibodies were at least 3-fold more potent in these types ofassays than the parental form, 10G5. An example of the fold-differencein functioning is illustrated in FIG. 11, where the EC₅₀s for 10G5 and10G5H6 were determined to be 2.9 μg/ml and 0.8 μg/ml, respectively, withIL-13 as the stimulant. FIG. 12 illustrates antibody inhibition of STAT6phosphorylation in NHDF cells, where IL-4 is used as the stimulant.Results of this analysis indicated EC₅₀s of 5.0 μg/ml and 1.8 μg/ml for10G5 and 10G5H6, respectively.

EXAMPLE 12 Functional Studies—TARC Release

Thymus and Activation-Regulated Chemokine (TARC) Release Assay (Dog,Rhesus or Human).

Blood was collected in heparinized VACUTAINER® tubes (VWR, VT6480).PBMCs were isolated over Lymphocyte Separation Media (ICN, 50494X).PBMCs or whole blood was plated in 96-well flat bottom plates (COSTAR,#2595). Antibodies were added and incubated for 30 minutes at roomtemperature. Recombinant rhesus IL-13 was added at 10 ng/ml finalconcentration and incubated for 24-72 hours at 37° C. with CO₂ in ahumidified chamber. Supernate or plasma was collected (TARC can bedetected as early as 24 hours but levels continue to increase). TARC wasmeasured by immunoassay. IMMULON®-4 plates (DYNATECH, 3855) were coatedwith anti-human TARC (R&D, #AF364) at 2 μg/ml in PBS (INVITROGEN,#14290-144), 50 μl/well. The plates were incubated overnight at 4° C.Blocking buffer (200 μl/well) was added and incubated for 1 hour at roomtemperature. The plates were washed three times with wash buffer. Plasmaor supernate was added, 50 μl/well, and incubated for 2 hours at roomtemperature (plasma diluted 1:2). A standard curve was included startingat 20 ng/ml recombinant human TARC diluted 2-fold. The plates werewashed three times with wash buffer. Detection was carried out withbiotin anti-human TARC (RDI, #RDI-TarcabrP1, conjugated to biotin 20:1molar ratio) at 250 ng/ml in blocking buffer (60 μl/well) for 1 hour atroom temperature. The plates were washed three times with wash buffer.Streptavidin-Europium (Wallac, #1244-360) was added 100 μl/well at 100ng/ml in europium buffer for 20 minutes at room temperature. The plateswere washed three times with wash buffer. Enhancement solution (Wallac,#12244-105), 150 μl/well, was added and incubated for 1 hour at roomtemperature. Time-delayed fluorescence was read in a VICTOR(PERKIN-ELMER) reader.

Results.

The effect of the present antibodies on TARC release followingstimulation with 10 ng/mL of IL-13 was examined. FIG. 13 illustrates,for example, the ability of 10G5-6 to block the IL-13-stimulated releaseof TARC (CCL17) in whole human blood. The antibody yielded an IC₅₀ of˜112 ng/mL, 746 pM. FIG. 14 illustrates functioning of another antibody,10G5H6, versus 10G5 WT (wild-type) in an IL-13-stimulated TARC releaseassay from whole human blood. FIG. 15 illustrates the effect of 10G5-6alongside 10G5H6 in blocking the release of TARC after stimulation ofwhole rhesus blood with rhesus IL-13.

EXAMPLE 13 Functional Studies—Inhibition of Cell Proliferation ofHodgkin's Disease

Methods.

10G5, 10G5H6 and 10G5-6 were assayed to determine whether the antibodieswere effective in inhibiting cell proliferation of Hodgkin's diseasecell line L1236. Hodgkin's and Reed-Sternberg cells have been studiedpreviously with regard to the cytokine IL-13; see, e.g., Kapp et al.,1999 J. Exp. Med. 189:1939-1945; Skinnider et al., 2001 Blood97:250-255; Skinnider et al., 2002 Blood 99:618-626; and in U.S. Pat.No. 6,468,528, and the methods of analyzing same are discussed therein.

Results.

In this assay, 10G5 gave an IC₅₀ of about 300 ng/mL (2 nM), whereas both10G5H6 and 10G5-6 yielded IC₅₀s of about 50 ng/ml (˜330 pM) which forboth represented a ca. 6-fold improvement over that of the parent 10G5IgG. See FIG. 17.

What is claimed is:
 1. An isolated antibody that binds to humaninterleukin 13 receptor alpha 1 and competes for binding to the receptorwith an antibody comprising: (a) a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 amino acid sequences as set forth in SEQID NO:82, SEQ ID NO:83, and SEQ ID NO:121, respectively; and (b) a lightchain variable region comprising CDR1, CDR2, and CDR3 amino acidsequences as set forth in SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:122,respectively, wherein said antibody does not bind human interleukin 13receptor alpha 1 comprising an alanine residue at position 233 of SEQ IDNO:101.
 2. A composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier.